Fluorescent activated cell sorting (FACS) is a specialized type of flow cytometry used for sorting and analyzing a heterogeneous mixture of cells into different subpopulations based on the specific light scattering and fluorescent characteristics (from the specific labels) of each cell.
This document provides an overview of flow cytometry, including its history, components, principles, and applications. Flow cytometry involves passing cells in suspension through a laser beam to measure physical properties like size and granularity, as well as cell markers detected by fluorescent antibodies. This allows identification of cell types, lineages, and abnormalities. The document discusses sample preparation, common specimens analyzed, immunophenotyping using multiple fluorochromes, and applications like DNA content analysis, erythrocyte analysis, and reticulocyte counting.
This document provides an introduction to flow cytometry. It defines flow cytometry as the measurement of physical and chemical characteristics of cells as they flow in a fluid stream through a beam of light. It describes the key components of a flow cytometer including fluidics to deliver cells to the laser, optics to excite and collect light, and electronics to amplify and process signals. It explains the different types of signals detected including light scatter and fluorescence, and how these can be used to characterize cells. The document provides guidance on choosing fluorochromes and considerations for multi-color panels such as spectral overlap. It outlines some common applications of flow cytometry and contact details.
Flow cytometry for cell componenet analysisRAJA GOPAL
Flow cytometry is a technique that uses lasers and fluorescence to analyze physical and chemical characteristics of cells as they flow in a fluid stream. It allows simultaneous analysis of thousands of cells per second based on parameters like cell size, granularity, and detection of cell surface antigens using specific antibodies labeled with fluorochromes of different colors. Specimens suitable for analysis include blood, bone marrow, body fluids, and cell suspensions generated from tissues. Flow cytometry has various applications like immunophenotyping, DNA analysis, diagnosis of conditions like PNH, reticulated cell counting, and blood bank testing.
FACS and MACS with their applications in biological research.Deepak Agarwal
Flow cytometry (FACS) and magnetic activated cell sorting (MACS) are techniques used to analyze and separate cells based on their physical and chemical characteristics. FACS uses lasers to detect cell properties and sort cells into containers one by one, while MACS uses magnetic microbeads attached to cells to separate them in high gradient magnetic fields. These techniques have various applications in research including identifying stem cells, characterizing cancer cells, studying cell cycles, and isolating cell populations for further analysis.
Flow cytometry works by passing cells in a fluid stream through a laser beam, which causes light scattering and fluorescence that is detected and analyzed. Cells are labeled with fluorescent markers and passed through the flow cell in a hydrodynamically focused stream. A laser excites the fluorescent molecules, which emit light at different wavelengths. Forward scatter detects cell size, side scatter detects internal complexity. Detectors convert light signals to digital data, which is analyzed through gating and dot plots to identify cell populations and properties. Flow cytometry allows rapid multi-parameter analysis of individual cells.
This document provides an overview of flow cytometry and fluorescence-activated cell sorting (FACS). It describes flow cytometry as a technique for measuring physical and chemical characteristics of cells as they flow in a fluid stream, allowing for single cell analysis. FACS extends this by using fluorescence to identify cell characteristics and sort cells into separate collections based on these characteristics. The key components of a flow cytometer are described as lasers, optics including filters and detectors, fluidics to hydrodynamically focus cells, and electronics to convert optical signals to digital data. Applications including cell phenotyping, apoptosis analysis, and cell cycle analysis are discussed. Cell sorting and quantitative analysis of cell cycle phases are also summarized.
This document provides an overview of flow cytometry, including its history, components, principles, and applications. Flow cytometry involves passing cells in suspension through a laser beam to measure physical properties like size and granularity, as well as cell markers detected by fluorescent antibodies. This allows identification of cell types, lineages, and abnormalities. The document discusses sample preparation, common specimens analyzed, immunophenotyping using multiple fluorochromes, and applications like DNA content analysis, erythrocyte analysis, and reticulocyte counting.
This document provides an introduction to flow cytometry. It defines flow cytometry as the measurement of physical and chemical characteristics of cells as they flow in a fluid stream through a beam of light. It describes the key components of a flow cytometer including fluidics to deliver cells to the laser, optics to excite and collect light, and electronics to amplify and process signals. It explains the different types of signals detected including light scatter and fluorescence, and how these can be used to characterize cells. The document provides guidance on choosing fluorochromes and considerations for multi-color panels such as spectral overlap. It outlines some common applications of flow cytometry and contact details.
Flow cytometry for cell componenet analysisRAJA GOPAL
Flow cytometry is a technique that uses lasers and fluorescence to analyze physical and chemical characteristics of cells as they flow in a fluid stream. It allows simultaneous analysis of thousands of cells per second based on parameters like cell size, granularity, and detection of cell surface antigens using specific antibodies labeled with fluorochromes of different colors. Specimens suitable for analysis include blood, bone marrow, body fluids, and cell suspensions generated from tissues. Flow cytometry has various applications like immunophenotyping, DNA analysis, diagnosis of conditions like PNH, reticulated cell counting, and blood bank testing.
FACS and MACS with their applications in biological research.Deepak Agarwal
Flow cytometry (FACS) and magnetic activated cell sorting (MACS) are techniques used to analyze and separate cells based on their physical and chemical characteristics. FACS uses lasers to detect cell properties and sort cells into containers one by one, while MACS uses magnetic microbeads attached to cells to separate them in high gradient magnetic fields. These techniques have various applications in research including identifying stem cells, characterizing cancer cells, studying cell cycles, and isolating cell populations for further analysis.
Flow cytometry works by passing cells in a fluid stream through a laser beam, which causes light scattering and fluorescence that is detected and analyzed. Cells are labeled with fluorescent markers and passed through the flow cell in a hydrodynamically focused stream. A laser excites the fluorescent molecules, which emit light at different wavelengths. Forward scatter detects cell size, side scatter detects internal complexity. Detectors convert light signals to digital data, which is analyzed through gating and dot plots to identify cell populations and properties. Flow cytometry allows rapid multi-parameter analysis of individual cells.
This document provides an overview of flow cytometry and fluorescence-activated cell sorting (FACS). It describes flow cytometry as a technique for measuring physical and chemical characteristics of cells as they flow in a fluid stream, allowing for single cell analysis. FACS extends this by using fluorescence to identify cell characteristics and sort cells into separate collections based on these characteristics. The key components of a flow cytometer are described as lasers, optics including filters and detectors, fluidics to hydrodynamically focus cells, and electronics to convert optical signals to digital data. Applications including cell phenotyping, apoptosis analysis, and cell cycle analysis are discussed. Cell sorting and quantitative analysis of cell cycle phases are also summarized.
Flow cytometry allows for the quantitative and qualitative analysis of cell properties as cells flow in a fluid stream through a laser. Cells are labeled with fluorescent markers and pass through the laser one by one. Light scattering and fluorescence emission are converted to digital signals which provide information on cell size, granularity, and marker expression. Data is displayed as histograms, dot plots, or density plots to identify cell populations and phenotypes.
Principle and applications of flow cytometryDinesh Gangoda
Flow cytometry is a technique used to analyze physical and chemical characteristics of cells or particles in suspension as they flow in a fluid stream past a laser. It works by fluorescently labeling cells and components, then passing them in single file through a laser which detects scattered and fluorescent light. This allows for quantitative and qualitative analysis of cell populations. Properties like size, granularity, and fluorescence intensity can be measured. Main applications include immunophenotyping, cell sorting, cell cycle analysis, apoptosis analysis, and measuring intracellular calcium flux and cell proliferation in response to stimuli.
The document provides an overview of flow cytometry, including its history, principles, and applications. It discusses how flow cytometry allows for the measurement of cellular characteristics like fluorescence and light scattering at high speeds. Key developments include the first apparatus for detecting bacteria in a fluid stream in 1947 and the first cell sorter in 1965. The term "fluorescence activated cell sorter" or FACS was coined in 1972. Flow cytometry integrates technologies like lasers, optics, fluidics, and electronics to analyze individual cells and measure parameters such as cell size, granularity, and receptor expression. It has various applications in fields like immunology, genetics, and microbiology.
An introduction to flow cytometry- Ashwini.RAshwini R
The document provides an introduction to flow cytometry. It describes flow cytometry as a technique that allows simultaneous multiparametric analysis of physical and chemical characteristics of single cells suspended in a fluid stream. Key components of a flow cytometer include fluidics, optics, detectors, and electronics. Cells are hydrodynamically focused into a single file stream and pass through a laser beam, where light scattering and fluorescence emissions provide information about cellular properties. Photodetectors convert light signals into electrical pulses that are analyzed. Flow cytometry has various applications including immunophenotyping, cell sorting, DNA content analysis, and cell cycle/proliferation analysis.
Flow cytometry is a technique that uses lasers to illuminate single cell suspensions labeled with fluorescent markers as they flow through the instrument, generating signals from scattered and fluorescent light that can identify cell subsets and characteristics and be analyzed by computer to provide diagnostic information about cell populations. A flow cytometer consists of fluidic, optical, and electronic systems to transport cells to the laser beam, direct resulting light signals to detectors, and convert those signals into electronic data for analysis. Flow cytometry can identify cell types using fluorescent antibodies targeting antigens, analyze DNA content to detect cancer cell abnormalities, and examine the cell cycle to determine proliferation rates of malignant cells.
How to become a flow cytometry expert in 4 daysCJ Xia
In this how to become expert in application X series, Boster Bio presents the comprehensive information for you to get a head start on the subject. In this case, you will learn everything you need to know to get started on flow cytometry. You will learn how it works, how to choose the right antibody for FACS, how to gate, and many more.
Fluorescent activated cell sorting (FACS) is a specialized type of flow cytometry used for sorting and analyzing a heterogeneous mixture of cells into different sub- populations based on the specific light scattering and fluorescent characteristics (from the specific labels) of each cell. The number of measurable parameters that can be used by this technology to separate cell populations is immense – starting from simple surface immunophenotyping to metabolic functions, cell cycle status, redox state, and DNA content analysis to name a few.
Since its inception, FACS has been used extensively in biomedical research and clinical diagnostics and therapeutics. The most common usage of FACS is seen in:
Analysis of whole human blood for diagnosing diseases, immunophenotyping
Sorting different blood cell fractions for ex-vivo manipulations and/or transplantations
Immuno-phenotypic analysis of murine blood to identify transgenic/knockout animals
Sorting and analysis of a slew of cell lines for various biological assays
Characterization and isolation of rare cells types like adult stem cells and cancer initiating cells
This document discusses methods for evaluating the cytotoxicity of nanoparticles. It describes several common cytotoxicity assays including MTT, WST, trypan blue exclusion, and assays using dehydrogenases. The MTT assay measures mitochondrial activity and is widely used. WST assays use water-soluble reagents and do not require crystal solubilization. Dehydrogenase assays offer high sensitivity by measuring multiple cell elements. The document also provides examples of studies that used these assays to evaluate the cytotoxicity of silver nanoparticles, magnetic nanoparticles, and other nanomaterials.
This document provides an overview of flow cytometry. It begins by defining flow cytometry as a technique for quantitative single cell analysis that counts, examines, and sorts cells based on optical properties like light scattering and fluorescence. It then describes the basic principles, components, and working of a flow cytometer. Key components include lasers, optical filters, and detectors. Cells in suspension pass through the laser one by one, with signals detected by photodiodes and photomultiplier tubes. Applications discussed include cell sorting, apoptosis analysis using markers like Annexin V and PI, and cell cycle analysis using DNA binding dyes or BrdU incorporation. Clinical uses involve hematologic malignancy diagnosis, residual disease detection, and monitoring treatments.
The document provides an overview of the basic principles and components of flow cytometry. It discusses how flow cytometry works by measuring the properties of cells in fluid flow, using a combination of fluidics to introduce cells, optics to generate and collect light signals, and electronics to convert signals to digital data. Key aspects summarized include how cells are hydrodynamically focused and interrogated by light scatter and fluorescence to derive information on their size, granularity, and marker expression that can be analyzed using software.
This document provides an overview of flow cytometry including:
- An introduction to flow cytometry techniques and applications from multiple speakers
- Descriptions of key components and parameters measured in flow cytometry like scatter, fluorescence, and fluorochromes
- Examples of flow cytometry applications in fields like cell viability, proliferation, and surface marker analysis
- A discussion of antibody conjugation methods and considerations for multi-color flow cytometry experiments
Flow cytometry (FCM) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles. In this process, a sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument.
Cell sorting is a technique used to separate specific cell populations from tissues. The first step involves disrupting connections between cells using enzymes or calcium-chelating agents. This converts the tissue into single cells. Flow cytometry can then identify and separate different cell types by measuring light scattering or fluorescence emitted from individually labeled cells as they pass through a laser. Specific cells are labeled with fluorescent antibody markers and can then be isolated from unlabeled cells using a fluorescence activated cell sorter.
What are stem cells? This presentation provides an overview of multiple different stem cells including embryonic stem cells, mesenchymal stem cells, cancer stem cells, induced pluripotent stem cells, hematopoietic stem cells and neural stem cells.
This document provides an introduction to flow cytometry. It defines flow cytometry as a method for sensing individual cells in a fluid stream as they pass through a laser beam, measuring light scattering and fluorescence. Key aspects of flow cytometry systems and methodology are described, including hydrodynamic focusing of cells, light scattering measurements, use of fluorescent markers, optical and electronic components, data acquisition and analysis techniques like gating and compensation. The history of technological developments in flow cytometry is also summarized.
Tissue microarrays allow high-throughput analysis of molecular targets in hundreds of tissue samples by extracting small cores from donor tissue blocks and re-embedding them into a single microarray block, preserving tissue for simultaneous analysis under uniform conditions while amplifying limited resources and decreasing costs compared to individual analyses; they can be used to study molecular changes in large cohorts retrospectively and prospectively for diagnostic, basic research, and drug discovery purposes; Creative Bioarray is highlighted as a source for pre-made and custom tissue microarrays with a large repository of human and animal samples and related pathological services.
A brief presentation on cell counting and cell viability assays. For cell cytotoxicity assays, you can check my profile where I have uploaded a separate file.
Prepared in July 2015
Flow cytometry is a method used for cell counting, cell sorting, biomarker detection and protein engineering. It uses lasers to enable simultaneous multiparametric analysis of the physical and chemical characteristics of up to thousands of particles per second. Cells must be suspended in a stream of fluid and incubated with fluorescent-labelled antibodies which detect the expression of cell surface and intracellular molecules. The suspension is then passed by an electronic detection apparatus. The protocol for this technique is similar to but differs from that used for indirect flow cytometry, aka fluorescent-activated cell sorting (FACS).
Immunofluorescence or Immunofluorescence Antibody Assay(IFA) is a traditional laboratory technique that utilizes fluorescent dyes to identify the presence of antibodies bound to specific antigens.
Immunofluorescence is a powerful technique which allows for the visualisation of proteins or antigens within a cell or a section of tissue. Utilizing the binding specificity of an antibody to a given antigen, antibodies chemically conjugated with fluorescent dyes can be used to bind to specific molecular targets. This allows for easy visualisation by confocal or fluorescent microscopy.
Flow cytometry allows for the quantitative and qualitative analysis of cell properties as cells flow in a fluid stream through a laser. Cells are labeled with fluorescent markers and pass through the laser one by one. Light scattering and fluorescence emission are converted to digital signals which provide information on cell size, granularity, and marker expression. Data is displayed as histograms, dot plots, or density plots to identify cell populations and phenotypes.
Principle and applications of flow cytometryDinesh Gangoda
Flow cytometry is a technique used to analyze physical and chemical characteristics of cells or particles in suspension as they flow in a fluid stream past a laser. It works by fluorescently labeling cells and components, then passing them in single file through a laser which detects scattered and fluorescent light. This allows for quantitative and qualitative analysis of cell populations. Properties like size, granularity, and fluorescence intensity can be measured. Main applications include immunophenotyping, cell sorting, cell cycle analysis, apoptosis analysis, and measuring intracellular calcium flux and cell proliferation in response to stimuli.
The document provides an overview of flow cytometry, including its history, principles, and applications. It discusses how flow cytometry allows for the measurement of cellular characteristics like fluorescence and light scattering at high speeds. Key developments include the first apparatus for detecting bacteria in a fluid stream in 1947 and the first cell sorter in 1965. The term "fluorescence activated cell sorter" or FACS was coined in 1972. Flow cytometry integrates technologies like lasers, optics, fluidics, and electronics to analyze individual cells and measure parameters such as cell size, granularity, and receptor expression. It has various applications in fields like immunology, genetics, and microbiology.
An introduction to flow cytometry- Ashwini.RAshwini R
The document provides an introduction to flow cytometry. It describes flow cytometry as a technique that allows simultaneous multiparametric analysis of physical and chemical characteristics of single cells suspended in a fluid stream. Key components of a flow cytometer include fluidics, optics, detectors, and electronics. Cells are hydrodynamically focused into a single file stream and pass through a laser beam, where light scattering and fluorescence emissions provide information about cellular properties. Photodetectors convert light signals into electrical pulses that are analyzed. Flow cytometry has various applications including immunophenotyping, cell sorting, DNA content analysis, and cell cycle/proliferation analysis.
Flow cytometry is a technique that uses lasers to illuminate single cell suspensions labeled with fluorescent markers as they flow through the instrument, generating signals from scattered and fluorescent light that can identify cell subsets and characteristics and be analyzed by computer to provide diagnostic information about cell populations. A flow cytometer consists of fluidic, optical, and electronic systems to transport cells to the laser beam, direct resulting light signals to detectors, and convert those signals into electronic data for analysis. Flow cytometry can identify cell types using fluorescent antibodies targeting antigens, analyze DNA content to detect cancer cell abnormalities, and examine the cell cycle to determine proliferation rates of malignant cells.
How to become a flow cytometry expert in 4 daysCJ Xia
In this how to become expert in application X series, Boster Bio presents the comprehensive information for you to get a head start on the subject. In this case, you will learn everything you need to know to get started on flow cytometry. You will learn how it works, how to choose the right antibody for FACS, how to gate, and many more.
Fluorescent activated cell sorting (FACS) is a specialized type of flow cytometry used for sorting and analyzing a heterogeneous mixture of cells into different sub- populations based on the specific light scattering and fluorescent characteristics (from the specific labels) of each cell. The number of measurable parameters that can be used by this technology to separate cell populations is immense – starting from simple surface immunophenotyping to metabolic functions, cell cycle status, redox state, and DNA content analysis to name a few.
Since its inception, FACS has been used extensively in biomedical research and clinical diagnostics and therapeutics. The most common usage of FACS is seen in:
Analysis of whole human blood for diagnosing diseases, immunophenotyping
Sorting different blood cell fractions for ex-vivo manipulations and/or transplantations
Immuno-phenotypic analysis of murine blood to identify transgenic/knockout animals
Sorting and analysis of a slew of cell lines for various biological assays
Characterization and isolation of rare cells types like adult stem cells and cancer initiating cells
This document discusses methods for evaluating the cytotoxicity of nanoparticles. It describes several common cytotoxicity assays including MTT, WST, trypan blue exclusion, and assays using dehydrogenases. The MTT assay measures mitochondrial activity and is widely used. WST assays use water-soluble reagents and do not require crystal solubilization. Dehydrogenase assays offer high sensitivity by measuring multiple cell elements. The document also provides examples of studies that used these assays to evaluate the cytotoxicity of silver nanoparticles, magnetic nanoparticles, and other nanomaterials.
This document provides an overview of flow cytometry. It begins by defining flow cytometry as a technique for quantitative single cell analysis that counts, examines, and sorts cells based on optical properties like light scattering and fluorescence. It then describes the basic principles, components, and working of a flow cytometer. Key components include lasers, optical filters, and detectors. Cells in suspension pass through the laser one by one, with signals detected by photodiodes and photomultiplier tubes. Applications discussed include cell sorting, apoptosis analysis using markers like Annexin V and PI, and cell cycle analysis using DNA binding dyes or BrdU incorporation. Clinical uses involve hematologic malignancy diagnosis, residual disease detection, and monitoring treatments.
The document provides an overview of the basic principles and components of flow cytometry. It discusses how flow cytometry works by measuring the properties of cells in fluid flow, using a combination of fluidics to introduce cells, optics to generate and collect light signals, and electronics to convert signals to digital data. Key aspects summarized include how cells are hydrodynamically focused and interrogated by light scatter and fluorescence to derive information on their size, granularity, and marker expression that can be analyzed using software.
This document provides an overview of flow cytometry including:
- An introduction to flow cytometry techniques and applications from multiple speakers
- Descriptions of key components and parameters measured in flow cytometry like scatter, fluorescence, and fluorochromes
- Examples of flow cytometry applications in fields like cell viability, proliferation, and surface marker analysis
- A discussion of antibody conjugation methods and considerations for multi-color flow cytometry experiments
Flow cytometry (FCM) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles. In this process, a sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument.
Cell sorting is a technique used to separate specific cell populations from tissues. The first step involves disrupting connections between cells using enzymes or calcium-chelating agents. This converts the tissue into single cells. Flow cytometry can then identify and separate different cell types by measuring light scattering or fluorescence emitted from individually labeled cells as they pass through a laser. Specific cells are labeled with fluorescent antibody markers and can then be isolated from unlabeled cells using a fluorescence activated cell sorter.
What are stem cells? This presentation provides an overview of multiple different stem cells including embryonic stem cells, mesenchymal stem cells, cancer stem cells, induced pluripotent stem cells, hematopoietic stem cells and neural stem cells.
This document provides an introduction to flow cytometry. It defines flow cytometry as a method for sensing individual cells in a fluid stream as they pass through a laser beam, measuring light scattering and fluorescence. Key aspects of flow cytometry systems and methodology are described, including hydrodynamic focusing of cells, light scattering measurements, use of fluorescent markers, optical and electronic components, data acquisition and analysis techniques like gating and compensation. The history of technological developments in flow cytometry is also summarized.
Tissue microarrays allow high-throughput analysis of molecular targets in hundreds of tissue samples by extracting small cores from donor tissue blocks and re-embedding them into a single microarray block, preserving tissue for simultaneous analysis under uniform conditions while amplifying limited resources and decreasing costs compared to individual analyses; they can be used to study molecular changes in large cohorts retrospectively and prospectively for diagnostic, basic research, and drug discovery purposes; Creative Bioarray is highlighted as a source for pre-made and custom tissue microarrays with a large repository of human and animal samples and related pathological services.
A brief presentation on cell counting and cell viability assays. For cell cytotoxicity assays, you can check my profile where I have uploaded a separate file.
Prepared in July 2015
Flow cytometry is a method used for cell counting, cell sorting, biomarker detection and protein engineering. It uses lasers to enable simultaneous multiparametric analysis of the physical and chemical characteristics of up to thousands of particles per second. Cells must be suspended in a stream of fluid and incubated with fluorescent-labelled antibodies which detect the expression of cell surface and intracellular molecules. The suspension is then passed by an electronic detection apparatus. The protocol for this technique is similar to but differs from that used for indirect flow cytometry, aka fluorescent-activated cell sorting (FACS).
Immunofluorescence or Immunofluorescence Antibody Assay(IFA) is a traditional laboratory technique that utilizes fluorescent dyes to identify the presence of antibodies bound to specific antigens.
Immunofluorescence is a powerful technique which allows for the visualisation of proteins or antigens within a cell or a section of tissue. Utilizing the binding specificity of an antibody to a given antigen, antibodies chemically conjugated with fluorescent dyes can be used to bind to specific molecular targets. This allows for easy visualisation by confocal or fluorescent microscopy.
Indirect flow cytometry, also known as fluorescence-activated cell sorting (FACS) is a specialised type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. It is a useful scientific instrument as it provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest.
In contrast to direct flow cytometry, FACS requires two incubation steps. Firstly with the appropriate primary antibody, then with a fluorochrome-labelled secondary antibody.
This method is used to visualise the localisation and quantity of a protein of interest. The target protein is bound to by a specific primary antibody, which in turn is detected by a secondary antibody conjugated to a fluorophore. A fluorescent or confocal microscope is used to visualise the protein.
Immunocytochemistry (ICC) differs from immunohistochemistry (IHC) in that the former is performed on samples of intact cells that have had most, if not all, of their surrounding extracellular matrix removed. In contrast, immunohistochemical samples are sections of biological tissue, where each cell is surrounded by tissue architecture and other cells normally found in the intact tissue. These differences cause the samples to be prepared differently. For ICC, the sample requires permeabilisation so that the antibodies can reach the intracellular targets. Depending on the thickness of the sample, IHC samples do not require this.
Do you have a technical question? Get in touch: info@stjohnslabs.com
The document describes protocols for preparing competent E. coli cells, transforming those cells with plasmid DNA, growing cultures of E. coli containing plasmids, and purifying plasmid DNA from the bacterial cells. Specifically, it provides detailed multi-step protocols for making competent cells, transforming the cells, preparing glycerol stocks and stab cultures for long-term storage of bacterial strains, recovering single colonies, monitoring bacterial growth, and lysing the bacterial cells to release plasmid DNA.
The student worked on evaluating antitumor agents in vitro through cell culture and MTT assays. For cell culture, the student subcultured adherent A549 cells and suspension Raji cells, and resuscitated frozen cells. For MTT assays, the student tested the antitumor drug BEZ235 on A549 cells at different concentrations and measured optical density values. Though the results were abnormal, the student gained experience in carrying out cell culture techniques and MTT assay procedures.
This document summarizes a study evaluating the efficacy of differentiating mouse embryonic stem cells (mESCs) into hepatocyte-like cells. The researchers found that cell cycle synchronization of mESCs prior to differentiation led to decreased expression of the pluripotency marker Oct4 and increased expression of endoderm and hepatic progenitor markers, indicating improved differentiation. Assays of stem cell-derived hepatocyte-like cells showed production of the liver proteins albumin and urea, though urea levels decreased over time, suggesting further optimization is needed to generate fully functional hepatocytes from mESCs. The researchers concluded cell cycle synchronization enhanced differentiation but that identifying additional progenitor factors could improve hepatic function of the derived cells.
This document summarizes an optimization project to screen lectins for their ability to capture pathogens. The author developed an optimized fixation protocol for pathogens and tested it using crystal violet staining and ELISA assays. ELISA results showed the protocol worked and FcMBL detected E. coli within detectable limits. Other lectins were screened and compared to FcMBL using ELISA. While results were promising, more optimization is needed regarding blocking agents and lectin concentrations. The author acknowledges contributions from mentors and colleagues.
Hematopoietic development of human embryonic stem cellsqussai abbas
This document discusses culturing and differentiation of human embryonic stem cells (hESCs). Key points include:
- hESCs can be maintained in an undifferentiated state for long periods in culture and differentiated into specific lineages.
- Induced pluripotent stem cells (iPSCs) provide an alternative to hESCs and can be generated from adult cells through nuclear reprogramming.
- hESCs are cultured on mouse embryonic fibroblast feeder layers and specific markers are used to identify undifferentiated hESCs. Differentiation can be induced through embryoid body formation or coculture with stromal cells to generate hematopoietic progenitors.
Hematopoietic development of human embryonic stem cellsQussai Abbas
Hematopoietic development of human embryonic stem cells
CULTURING HUMAN EMBRYONIC STEM CELLS
Hematopoietic differentiation of HUMAN EMBRYONIC STEM CELLS
S17 stromal cells
Embryoid bodies
Dna extraction from fresh or frozen tissuesCAS0609
This document provides a protocol for extracting DNA from fresh or frozen tissues. The protocol involves disaggregating tissue samples and lysing cells to release DNA. Proteins are then digested and DNA is separated from other cellular components using phenol/chloroform extraction. The extracted DNA is precipitated and purified by ethanol precipitation. DNA concentration and quality can be assessed using spectrophotometry and gel electrophoresis. The protocol notes that traditional organic extraction effectively isolates high molecular weight DNA but commercial non-organic kits provide a faster alternative while avoiding toxic phenol.
1. The document describes methods for stably transfecting cell lines, including calcium phosphate transfection, lipofection, and spontaneous transfection.
2. It examines the efficiency of transfection for different cell lines using green fluorescent protein and red fluorescent protein plasmids.
3. Results show that calcium phosphate transfection was more efficient than spontaneous transfection for most cell lines tested, except HeLa cells. Stable transfection of HeLa cells over 21 days resulted in gradually increasing fluorescent intensity that then remained constant.
This document provides protocols and reagents for performing western blotting, including lysis buffer, loading buffer, running buffer, transfer buffer, staining buffer, blocking buffer, and stripping buffer compositions. It describes sample preparation involving cell lysis and sonication. Protocols are provided for protein separation via SDS-PAGE gel electrophoresis, protein transfer to a membrane, antibody incubation, imaging, and stripping/reprobing the membrane. The goal is to detect target proteins on the membrane using chemiluminescent detection and normalization to loading control protein levels.
This document describes methods for stably transfecting cell lines to create new cell lines. It compares the calcium phosphate precipitation method, spontaneous transfection method, and lipofection method. It also details monitoring transfection efficiency by fluorescence microscopy and spectroscopy. Results show the calcium phosphate method worked best for most cell lines tested except HeLa cells. Stable transfection of HeLa cells with a plasmid increased fluorescence over 21 days then plateaued. Plasmid DNA concentration did not correlate with fluorescence yield.
This document describes methods for stably transfecting cell lines to create new cell lines. It compares the calcium phosphate precipitation method, spontaneous transfection method, and lipofection method. It also details monitoring transfection efficiency by fluorescence microscopy and spectroscopy. Results show the calcium phosphate method worked best for most cell lines tested except HeLa cells. Stable transfection of HeLa cells with a plasmid increased fluorescence over 21 days then plateaued. Plasmid DNA concentration did not correlate with fluorescence yield.
This document describes a quantitative ELISA assay for detecting fumonisin levels in urine samples. Fumonisins are mycotoxins produced by fungi that have been linked to various cancers and diseases. The assay uses antibody-coated microwells to competitively bind fumonisin from urine samples and an HRP-conjugated detection antibody. A colorimetric readout is used to quantify fumonisin levels, which are interpolated from a standard curve. The procedure involves purifying fumonisin from urine samples using a cleanup column before performing the ELISA assay to achieve quantitative results.
The complete guide to antibody detection by immunofluorescence techniqueCandy Swift
The document provides instructions for using immunofluorescence techniques to detect antibodies. It describes how to prepare cell samples and reagents, and outlines two main methods - indirect immunofluorescence and cell membrane fluorescence staining. Indirect immunofluorescence involves incubating cell samples with antibody samples to be tested, then a fluorescent secondary antibody, and viewing under a microscope. Cell membrane fluorescence staining uses living cell suspensions incubated at 4°C with antibody samples and fluorescent antibodies to stain just the cell membrane.
This document provides information on various techniques for disaggregating animal tissue into single cells for cell culture, including mechanical, enzymatic, warm trypsinization, cold trypsinization, and using collagenase. It also discusses subculturing cells, cell counting using a hemocytometer, viability assays with trypan blue staining, maintaining aseptic technique to prevent contamination, and cryopreserving cells in liquid nitrogen with DMSO. The key steps and principles of each technique are explained.
Cells were thawed and plated, then trypsinized and passaged to detach and transfer cells to new plates. Cells were quantified using a hemocytometer after staining with trypan blue. Around 58% viability was observed. Cells were then cryopreserved in DMSO for storage in liquid nitrogen. Proper techniques like quick thawing, plating in fresh media, and passaging help keep cells alive through multiple procedures in cell culture work.
growth and maintenence of plant tissue culturemarymelna1
This document discusses the establishment and maintenance of plant tissue cultures, including callus culture and suspension culture. It provides details on the initiation and various growth phases of callus culture and suspension culture. The key steps for callus culture include selection of explant, preparation of culture medium, transfer of explant, and incubation. For suspension culture, callus fragments or single cells are transferred to liquid medium with agitation to keep cells separate. The growth of plant tissue cultures can be determined through methods like cell counting, packed cell volume, fresh cell weight, and viable cell tests. Subculturing is needed every 4-5 weeks to maintain callus growth due to nutrient depletion and toxin accumulation in the medium.
Similar to FACS Sample Preparation & Protocol (20)
Does your next experiment involve Leptin (LEP)? This is a presentation about LEP intended for scientists who are designing controls and performing immunoassays detecting LEP. It contains useful info such as Western blot band size, protein expression, and interesting facts.
Anti-Leptin Antibody Picoband™ (A00479-3):
https://www.bosterbio.com/anti-leptin-picoband-trade-antibody-a00479-3-boster.html
References: Uniprot.org, ProteinAtlas.org, PMC2970652, PMC7520685, PMC3033396
Learn more about LEP (infographic and discussion): https://www.bosterbio.com/bosterbio-gene-info-cards/LEP
Boster Biological Technology
Website: www.bosterbio.com
Email: support@bosterbio.com
Does your next experiment involve Fibronectin (FN)? This is a presentation about FN1 intended for scientists who are designing controls and performing immunoassays detecting FN1. It contains useful info such as Western blot band size, protein expression, and interesting facts.
Anti-Fibronectin/FN1 Antibody Picoband™ (A00564-1): https://www.bosterbio.com/anti-fibronectin-fn1-picoband-trade-antibody-a00564-1-boster.html
References: Uniprot.org, ProteinAtlas.org, GeneCards, PMC5112592, PMC3071080,PMC6073216, PMC6204085
Learn more about FN1 (infographic and discussion): https://www.bosterbio.com/bosterbio-gene-info-cards/FN1
Boster Biological Technology
Website: www.bosterbio.com
Email: support@bosterbio.com
Does your next experiment involve Fibroblast growth factor 23 (FGF23)? This is a presentation about FGF23 intended for scientists who are designing controls and performing immunoassays detecting FGF23. It contains useful info such as Western blot band size, protein expression, and interesting facts.
Anti-FGF23 Antibody Picoband™ (PB9868): https://www.bosterbio.com/anti-fgf23-picoband-trade-antibody-pb9868-boster.html
References: Uniprot.org, ProteinAtlas.org, PMC5595838, PMC7010663, PMC7880350, PMC4121311
Learn more about FGF23 (infographic and discussion): https://www.bosterbio.com/bosterbio-gene-info-cards/FGF23
Boster Biological Technology
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Does your next experiment involve Fms-related tyrosine kinase 1 (FLT1)? This is a presentation about FLT1/VEGFR1 intended for scientists who are designing controls and performing immunoassays detecting FLT1/VEGFR1. It contains useful info such as Western blot band size, protein expression, and interesting facts.
Anti-FLT1 Antibody Picoband™ (A00534-4): https://www.bosterbio.com/anti-flt1-picoband-trade-antibody-a00534-4-boster.html
References: Uniprot.org, ProteinAtlas.org, PMC6594043, PMC4710184, PMC2615562
Learn more about FLT1/VEGFR1 (infographic and discussion): https://www.bosterbio.com/bosterbio-gene-info-cards/FLT1
Boster Biological Technology
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Does your next experiment involve Plasminogen Activator, Urokinase Receptor (PLAUR)? This is a presentation about uPAR/PLAUR intended for scientists who are designing controls and performing immunoassays detecting uPAR (Urokinase plasminogen activator surface receptor). It contains useful info such as Western blot band size, protein expression, and interesting facts
Anti-UPA Receptor/Plaur Antibody Picoband™ (A00993-2):
https://www.bosterbio.com/anti-upa-receptor-picoband-trade-antibody-a00993-2-boster.html
References: Uniprot.org, ProteinAtlas.org, PMC2586916, PMC6653070, PMC4159539, PMC6355443
Learn more about uPAR/PLAUR (infographic and discussion):
https://www.bosterbio.com/bosterbio-gene-info-cards/PLAUR
Boster Biological Technology
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Does your next experiment involve Chemokine (C-X-C motif) ligand 13 (CXCL13)? This is a presentation about CXCL13/BCA1/BLC intended for scientists who are designing controls and performing immunoassays detecting CXCL13/BCA1/BLC. It contains useful info such as Western blot band size, protein expression, and interesting facts.
Anti-BCA1/CXCL13 Antibody Picoband™ (PB9999): https://www.bosterbio.com/anti-bca1-picoband-trade-antibody-pb9999-boster.html
References: Uniprot.org, ProteinAtlas.org, GeneCards, PMC5307320, PMC7422843, PMC4940825
Learn more about CXCL13 (infographic and discussion): https://www.bosterbio.com/bosterbio-gene-info-cards/CXCL13
Boster Biological Technology
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Does your next experiment involve Integrin alpha-3? This is a presentation about Integrin alpha 3/ITGA3 intended for scientists who are designing controls and performing immunoassays detecting Integrin alpha 3/ITGA3. It contains useful info such as Western blot band size, protein expression, and interesting facts.
Anti-Integrin Alpha 3/ITGA3 Antibody (PA1621):
https://www.bosterbio.com/anti-integrin-alpha-3-antibody-pa1621-boster.html
References: Uniprot.org, ProteinAtlas.org, PMC6034741, PMC2947217, PMC7327512
Learn more about Integrin alpha 3/ITGA3 (infographic and discussion): https://www.bosterbio.com/bosterbio-gene-info-cards/ITGA3
Boster Biological Technology
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Does your next experiment involve Chemokine (C-C motif) ligand 2 (CCL2)? This is a presentation about MCP-1/CCL2 intended for scientists who are designing controls and performing immunoassays detecting MCP-1/CCL2. It contains useful info such as Western blot band size, protein expression, and interesting facts.
Anti-MCP1/CCL2 Antibody Picoband™ (PB9570): https://www.bosterbio.com/anti-mcp-1-picoband-trade-antibody-pb9570-boster.html
References: Uniprot.org, ProteinAtlas.org, PMC5261840, PMC8854715, PMC6883042, PMC5428676
Learn more about MCP-1/CCL2 (infographic and discussion): https://www.bosterbio.com/bosterbio-gene-info-cards/CCL2
Boster Biological Technology
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Does your next experiment involve Insulin-like growth factor I (IGF-I, IGF-1)? This is a presentation about IGF1 intended for scientists who are designing controls and performing immunoassays detecting IGF1. It contains useful info such as Western blot band size, protein expression, and interesting facts.
Anti-IGF1 Mouse Monoclonal Antibody (M00148-1): https://www.bosterbio.com/anti-igf1-mouse-monoclonal-antibody-clone-id-oti4b12-m00148-1-boster.html
References: Uniprot.org, ProteinAtlas.org, PMC5867890, PMC7450330, PMC7262660, PMC7401641
Learn more about IGF1 (infographic and discussion): https://www.bosterbio.com/bosterbio-gene-info-cards/IGF1
Boster Biological Technology
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Does your next experiment involve Periostin (POSTN)? This is a presentation about POSTN intended for scientists who are designing controls and performing immunoassays detecting POSTN. It contains useful info such as Western blot band size, protein expression, and interesting facts.
Anti-Periostin/Postn Antibody Picoband™ (A01378):
https://www.bosterbio.com/anti-periostin-picoband-trade-antibody-a01378-boster.html
References: Uniprot.org, ProteinAtlas.org, PMC4826188, PMC4746667, PMC4262539, PMC3443161
Learn more about POSTN (infographic and discussion): https://www.bosterbio.com/bosterbio-gene-info-cards/POSTN
Boster Biological Technology
Website: www.bosterbio.com
Email: support@bosterbio.com
Does your next experiment involve Angiotensin-converting enzyme (ACE)? This is a presentation about ACE intended for scientists who are designing controls and performing immunoassays detecting ACE. It contains useful info such as Western blot band size, protein expression, and interesting facts.
Anti-Angiotensin Converting Enzyme 1/ACE Antibody (PA2196-2): https://www.bosterbio.com/anti-angiotensin-converting-enzyme-1-antibody-pa2196-2-boster.html
References: Uniprot.org, ProteinAtlas.org, PMC3322615, PMC5722543, PMC6672927, PMC3389005
Learn more about ACE (infographic and discussion): https://www.bosterbio.com/bosterbio-gene-info-cards/ACE
Boster Biological Technology
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Email: support@bosterbio.com
Does your next experiment involve Lipopolysaccharide binding protein (LBP)? This is a presentation about LBP intended for scientists who are designing controls and performing immunoassays detecting LBP. It contains useful info such as Western blot band size, protein expression, and interesting facts.
Anti-LBP Antibody Picoband™ (A00809-1):
https://www.bosterbio.com/anti-lbp-picoband-trade-antibody-a00809-1-boster.html
References: Uniprot.org, ProteinAtlas.org, PMC3617275, PMC3485520, PMC6945416, PMC8710911
Learn more about LBP (infographic and discussion): https://www.bosterbio.com/bosterbio-gene-info-cards/LBP
Boster Biological Technology
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Does your next experiment involve NGFβ? This is a presentation about NGF/NGF Beta intended for scientists who are designing controls and performing immunoassays detecting NGF/beta-NGF. It contains useful info such as Western blot band size, protein expression, and interesting facts.
Anti-NGF/NGF Beta Antibody Picoband™ (A00341): https://www.bosterbio.com/anti-ngf-ngf-beta-picoband-trade-antibody-a00341-boster.html
References: Uniprot.org, ProteinAtlas.org, GeneCards, PMC4760111, PMC6037903, PMC2650228, PMC1894684
Learn more about NGF/NGF Beta (infographic and discussion): https://www.bosterbio.com/bosterbio-gene-info-cards/NGF
Boster Biological Technology
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Does your next experiment involve CD133 (Prominin-1)? This is a presentation about CD133/Prominin-1/PROM1 intended for scientists who are designing controls and performing immunoassays detecting Prominin-1. It contains useful info such as Western blot band size, protein expression, and interesting facts.
Anti-PROM1 Antibody Picoband™ (PB9156):
https://www.bosterbio.com/anti-prom1-picoband-trade-antibody-pb9156-boster.html
References: Uniprot.org, ProteinAtlas.org, PMC6763460, PMC2713505, PMC6030467
Learn more about CD133/Prominin-1/PROM1 (infographic and discussion): https://www.bosterbio.com/bosterbio-gene-info-cards/PROM1
Boster Biological Technology
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Does your next experiment involve CSF3? This is a presentation about CSF3/G-CSF intended for scientists who are designing controls and performing immunoassays detecting CSF3/G-CSF. It contains useful info such as Western blot band size, protein expression, and interesting facts.
Anti-G-CSF CSF3 Rabbit Monoclonal Antibody (M02280-1):
https://www.bosterbio.com/anti-g-csf-rabbit-monoclonal-antibody-m02280-1-boster.html
Anti-G-CSF/CSF3 Antibody Picoband™ (PB9563):
https://www.bosterbio.com/anti-g-csf-picoband-trade-antibody-pb9563-boster.html
References: Uniprot.org, ProteinAtlas.org, GeneCards, PMC5758815, PMC7196588, PMC8086136
Learn more about CSF3/G-CSF (infographic and discussion): https://www.bosterbio.com/bosterbio-gene-info-cards/CSF3
Boster Biological Technology
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Does your next experiment involve RANKL? This is a presentation about RANKL/TRANCE intended for scientists who are designing controls and performing immunoassays detecting TNFSF11/RANKL/TRANCE. It contains useful info such as Western blot band size, protein expression, and interesting facts.
Anti-RANKL/TNFSF11 Antibody Picoband™ (PB10015):
https://www.bosterbio.com/anti-rankl-picoband-trade-antibody-pb10015-boster.html
References: Uniprot.org, ProteinAtlas.org, GeneCards, PMC8602693, PMID 29203513, PMC7949628
Learn more about TNFSF11/RANKL/TRANCE/OPGL/ODF (infographic and discussion): https://www.bosterbio.com/bosterbio-gene-info-cards/TNFSF11
Boster Biological Technology
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Does your next experiment involve Neurofilament Heavy Polypeptide (NEFH)? This is a presentation about NEFH/NF-H intended for scientists who are designing controls and performing immunoassays detecting NEFH/NF-H. It contains useful info such as Western blot band size, protein expression, and interesting facts.
Anti-NEFH/Nf H Rabbit Monoclonal Antibody (M05307-2):
https://www.bosterbio.com/anti-nefh-rabbit-monoclonal-antibody-m05307-2-boster.html
References: Uniprot.org, ProteinAtlas.org, PMC6834541, PMC6054723, PMC4285109
Learn more about NEFH/NF-H (infographic and discussion): https://www.bosterbio.com/bosterbio-gene-info-cards/NEFH
Boster Biological Technology
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Does your next experiment involve Kidney injury molecule 1 (KIM-1)? This is a presentation about KIM1/TIM1/HAVCR1 intended for scientists who are designing controls and performing immunoassays detecting KIM-1/TIM-1/HAVCR1. It contains useful info such as Western blot band size, protein expression, and interesting facts.
Anti-TIM 1/HAVCR1 Antibody (PA1624): https://www.bosterbio.com/anti-tim-1-antibody-pa1624-boster.html
References: Uniprot.org, ProteinAtlas.org, PMC4552842, PMC8134587, PMC8139578, PMC7414978
Learn more about KIM-1/TIM-1/HAVCR1 (infographic and discussion): https://www.bosterbio.com/bosterbio-gene-info-cards/HAVCR1
Boster Biological Technology
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Does your next experiment involve Interferon gamma? This is a presentation about IFN Gamma/IFNG intended for scientists who are designing controls and performing immunoassays detecting IFN Gamma/IFNG. It contains useful info such as Western blot band size, protein expression, and interesting facts.
Anti-Interferon Gamma IFNG Rabbit Monoclonal Antibody (M00393-1): https://www.bosterbio.com/anti-interferon-gamma-rabbit-monoclonal-antibody-m00393-1-boster.html
Anti-IFN Gamma Antibody (RP1001):
https://www.bosterbio.com/anti-rat-ifn-gamma-antibody-rp1001-boster.html
References: Uniprot.org, ProteinAtlas.org, PMID15220936, PMID9524237, PMID24253448, PMC7086207
Learn more about IFN Gamma/IFNG (infographic and discussion): https://www.bosterbio.com/bosterbio-gene-info-cards/IFNG
Boster Biological Technology
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Email: support@bosterbio.com
Does your next experiment involve Bone morphogenetic protein 2 (BMP-2)? This is a presentation about BMP-2 intended for scientists who are designing controls and performing immunoassays detecting BMP-2. It contains useful info such as Western blot band size, protein expression, and interesting facts.
Anti-BMP2 Rabbit Polyclonal Antibody (A00338): https://www.bosterbio.com/anti-bmp2-rabbit-polyclonal-antibody-a00338-boster.html
References: Uniprot.org, ProteinAtlas.org, PMC5645599, PMC8082363, PMC5425130, PMC8170469
Learn more about BMP-2 (infographic and discussion): https://www.bosterbio.com/bosterbio-gene-info-cards/BMP2
Boster Biological Technology
Website: www.bosterbio.com
Email: support@bosterbio.com
The cost of acquiring information by natural selectionCarl Bergstrom
This is a short talk that I gave at the Banff International Research Station workshop on Modeling and Theory in Population Biology. The idea is to try to understand how the burden of natural selection relates to the amount of information that selection puts into the genome.
It's based on the first part of this research paper:
The cost of information acquisition by natural selection
Ryan Seamus McGee, Olivia Kosterlitz, Artem Kaznatcheev, Benjamin Kerr, Carl T. Bergstrom
bioRxiv 2022.07.02.498577; doi: https://doi.org/10.1101/2022.07.02.498577
EWOCS-I: The catalog of X-ray sources in Westerlund 1 from the Extended Weste...Sérgio Sacani
Context. With a mass exceeding several 104 M⊙ and a rich and dense population of massive stars, supermassive young star clusters
represent the most massive star-forming environment that is dominated by the feedback from massive stars and gravitational interactions
among stars.
Aims. In this paper we present the Extended Westerlund 1 and 2 Open Clusters Survey (EWOCS) project, which aims to investigate
the influence of the starburst environment on the formation of stars and planets, and on the evolution of both low and high mass stars.
The primary targets of this project are Westerlund 1 and 2, the closest supermassive star clusters to the Sun.
Methods. The project is based primarily on recent observations conducted with the Chandra and JWST observatories. Specifically,
the Chandra survey of Westerlund 1 consists of 36 new ACIS-I observations, nearly co-pointed, for a total exposure time of 1 Msec.
Additionally, we included 8 archival Chandra/ACIS-S observations. This paper presents the resulting catalog of X-ray sources within
and around Westerlund 1. Sources were detected by combining various existing methods, and photon extraction and source validation
were carried out using the ACIS-Extract software.
Results. The EWOCS X-ray catalog comprises 5963 validated sources out of the 9420 initially provided to ACIS-Extract, reaching a
photon flux threshold of approximately 2 × 10−8 photons cm−2
s
−1
. The X-ray sources exhibit a highly concentrated spatial distribution,
with 1075 sources located within the central 1 arcmin. We have successfully detected X-ray emissions from 126 out of the 166 known
massive stars of the cluster, and we have collected over 71 000 photons from the magnetar CXO J164710.20-455217.
Unlocking the mysteries of reproduction: Exploring fecundity and gonadosomati...AbdullaAlAsif1
The pygmy halfbeak Dermogenys colletei, is known for its viviparous nature, this presents an intriguing case of relatively low fecundity, raising questions about potential compensatory reproductive strategies employed by this species. Our study delves into the examination of fecundity and the Gonadosomatic Index (GSI) in the Pygmy Halfbeak, D. colletei (Meisner, 2001), an intriguing viviparous fish indigenous to Sarawak, Borneo. We hypothesize that the Pygmy halfbeak, D. colletei, may exhibit unique reproductive adaptations to offset its low fecundity, thus enhancing its survival and fitness. To address this, we conducted a comprehensive study utilizing 28 mature female specimens of D. colletei, carefully measuring fecundity and GSI to shed light on the reproductive adaptations of this species. Our findings reveal that D. colletei indeed exhibits low fecundity, with a mean of 16.76 ± 2.01, and a mean GSI of 12.83 ± 1.27, providing crucial insights into the reproductive mechanisms at play in this species. These results underscore the existence of unique reproductive strategies in D. colletei, enabling its adaptation and persistence in Borneo's diverse aquatic ecosystems, and call for further ecological research to elucidate these mechanisms. This study lends to a better understanding of viviparous fish in Borneo and contributes to the broader field of aquatic ecology, enhancing our knowledge of species adaptations to unique ecological challenges.
Current Ms word generated power point presentation covers major details about the micronuclei test. It's significance and assays to conduct it. It is used to detect the micronuclei formation inside the cells of nearly every multicellular organism. It's formation takes place during chromosomal sepration at metaphase.
The binding of cosmological structures by massless topological defectsSérgio Sacani
Assuming spherical symmetry and weak field, it is shown that if one solves the Poisson equation or the Einstein field
equations sourced by a topological defect, i.e. a singularity of a very specific form, the result is a localized gravitational
field capable of driving flat rotation (i.e. Keplerian circular orbits at a constant speed for all radii) of test masses on a thin
spherical shell without any underlying mass. Moreover, a large-scale structure which exploits this solution by assembling
concentrically a number of such topological defects can establish a flat stellar or galactic rotation curve, and can also deflect
light in the same manner as an equipotential (isothermal) sphere. Thus, the need for dark matter or modified gravity theory is
mitigated, at least in part.
The technology uses reclaimed CO₂ as the dyeing medium in a closed loop process. When pressurized, CO₂ becomes supercritical (SC-CO₂). In this state CO₂ has a very high solvent power, allowing the dye to dissolve easily.
Phenomics assisted breeding in crop improvementIshaGoswami9
As the population is increasing and will reach about 9 billion upto 2050. Also due to climate change, it is difficult to meet the food requirement of such a large population. Facing the challenges presented by resource shortages, climate
change, and increasing global population, crop yield and quality need to be improved in a sustainable way over the coming decades. Genetic improvement by breeding is the best way to increase crop productivity. With the rapid progression of functional
genomics, an increasing number of crop genomes have been sequenced and dozens of genes influencing key agronomic traits have been identified. However, current genome sequence information has not been adequately exploited for understanding
the complex characteristics of multiple gene, owing to a lack of crop phenotypic data. Efficient, automatic, and accurate technologies and platforms that can capture phenotypic data that can
be linked to genomics information for crop improvement at all growth stages have become as important as genotyping. Thus,
high-throughput phenotyping has become the major bottleneck restricting crop breeding. Plant phenomics has been defined as the high-throughput, accurate acquisition and analysis of multi-dimensional phenotypes
during crop growing stages at the organism level, including the cell, tissue, organ, individual plant, plot, and field levels. With the rapid development of novel sensors, imaging technology,
and analysis methods, numerous infrastructure platforms have been developed for phenotyping.
The use of Nauplii and metanauplii artemia in aquaculture (brine shrimp).pptxMAGOTI ERNEST
Although Artemia has been known to man for centuries, its use as a food for the culture of larval organisms apparently began only in the 1930s, when several investigators found that it made an excellent food for newly hatched fish larvae (Litvinenko et al., 2023). As aquaculture developed in the 1960s and ‘70s, the use of Artemia also became more widespread, due both to its convenience and to its nutritional value for larval organisms (Arenas-Pardo et al., 2024). The fact that Artemia dormant cysts can be stored for long periods in cans, and then used as an off-the-shelf food requiring only 24 h of incubation makes them the most convenient, least labor-intensive, live food available for aquaculture (Sorgeloos & Roubach, 2021). The nutritional value of Artemia, especially for marine organisms, is not constant, but varies both geographically and temporally. During the last decade, however, both the causes of Artemia nutritional variability and methods to improve poorquality Artemia have been identified (Loufi et al., 2024).
Brine shrimp (Artemia spp.) are used in marine aquaculture worldwide. Annually, more than 2,000 metric tons of dry cysts are used for cultivation of fish, crustacean, and shellfish larva. Brine shrimp are important to aquaculture because newly hatched brine shrimp nauplii (larvae) provide a food source for many fish fry (Mozanzadeh et al., 2021). Culture and harvesting of brine shrimp eggs represents another aspect of the aquaculture industry. Nauplii and metanauplii of Artemia, commonly known as brine shrimp, play a crucial role in aquaculture due to their nutritional value and suitability as live feed for many aquatic species, particularly in larval stages (Sorgeloos & Roubach, 2021).
2. Sample Preparation and Protocols
A. Preparation of Cells for FACS Staining
Cells for flow cytometry analysis are usually derived from four main sources:
- Adherent or suspension cultures
- Cryopreserved samples
- Whole blood or buffy coats
- Solid tissues e.g. bone marrow, spleen, intestine etc.
Regardless of the source, the final cell preparation should be:
- a homogenous single-cell suspension free of clumps and dead cell debris
- at a density of 106
-107
cell per ml
- suspended in a suitable staining buffer
Please see the following recommended protocols for sample preparation depending on your initial
sample source. PBMCs isolated from whole blood through Ficoll
19
3. gradient centrifugation or RBC lysed whole blood or non-adherent cultured cells are readily
available for flow cytometric analysis. Adherent cultured cells or cells present in the solid organs
should be first made into a single cell suspension before flow analysis by using enzymatic digestion
or mechanical dissociation of the tissue, respectively. Mechanical filtration should be followed to
avoid unwanted instrument clogs or lower quality flow data.
1.. Preparation of suspension culture cells Key
Reagents – PBS, staining buffer
1) Decant cells from tissue culture vessel into centrifuge tube(s).
2) Centrifuge at 300-400 x g for 5-10 min at room temperature.
3) Discard supernatant and re-suspend pellet in PBS and repeat previous step.
4) Discard supernatant and re-suspend in suitable volume of cold staining buffer.
2. . Preparation of adherent culture cells
Key Reagents – PBS, staining buffer, 0.25% trypsin
1) Discard culture medium and rinse the cell monolayer with sterile PBS
2) Add warmed trypsin to just cover the monolayer and incubate at 37°C for 5-10 min
(depending on the cell type).
3) Neutralize the reaction with culture medium (serum added) and detach the cells by gently
shaking the vessel.
4) Continue as with suspension culture cells preparation protocol.
3. . Preparation of cryopreserved cells
Key reagents – PBS, staining buffer, culture medium with 10% FBS
1) Thaw the cryo-tubes rapidly in a water bath set at 37°C.
2) Transfer to a chilled centrifuge tube and add ice cold culture medium drop by drop until the
cells are diluted 10X. Perform on ice!
3) Centrifuge at 300-400 x g for 5 min at 4°C.
4) Discard supernatant and wash once with cold staining buffer.
5) Re-suspend cells in a suitable volume of cold staining buffer.
4. . Preparation of Blood Mononuclear Cells (from blood or buffy coat)
Key reagents – PBS, staining buffer, suitable gradient medium like Ficoll or Histopaque
20
4. 5) To remove granulocytes, aspirate the whitish colored layer just above the RBC sediment
[Note: specialized gradient media have been formulated to enrich different granulocyte
populations].
6) Re-suspend the cells in PBS and centrifuge at 300-400 x g for 10 min. at room temperature.
7) Wash with PBS once or twice more.
8) Re-suspend the cells in a suitable volume of staining buffer.
5. . Preparation of cells from tissues - murine bone marrow
Key reagents – PBS, staining buffer, RBC lysis buffer [use common recipe or use a commercially
available buffer]
1) Dissect out the tibia and femurs and remove all extraneous fat, muscle and connective tissue.
Put the bone in cold PBS and store on ice – perform all following steps on ice.
2) Fill up a syringe with cold culture medium and fit an 18 gauge needle to it. Drill the ends of
the bones with the needle and flush out the contents onto a non- tissue culture treated plate.
3) Break up the cell masses into a single cell suspension with the help of a 22- gauge needle.
4) Pellet down the cells at 300-400 x g and wash once more with cold PBS.
5) Re-suspend the cells in a suitable volume of staining buffer and perform a cell count before
pelleting the cells again.
6) Re-suspend the cells in RBC lysis buffer at 106
cells/ml and incubate at room temperature for
5 minutes.
7) Pellet down the cells, discard the lysis buffer and wash once with PBS.
8) Re-suspend the cells in a suitable volume of staining buffer.
B. Common FACS staining protocols
Each human cell expresses hundreds of thousands of cell surface antigens that specify their cell
type, biological function, development stage, and much more. Cells residing in different organs
have characteristic cell surface antigens, and determination of these cells using the specific
fluorophore-conjugated antibodies can be analysed by flow cytometry. The following general
protocols are recommended for various common FACS staining procedures. Staining with
unconjugated purified antibody needs an additional step of staining with a fluorescent conjugated
secondary antibody (indirect immunostaining).
Note: If the cells are stained in a 96 well U- or V-bottom plate, washing procedure should be set up
first for maximum removal of unbound primary antibodies.
21
5. 2) Dispense 100µl of cell suspension into as many staining tubes as needed [unstained control,
compensation controls, optional isotype and FMO controls, and test sample].
3) Add the optimized dilution of antibodies to the respective tubes and incubate at 4°C (on ice)
for 30 minutes in the dark.
4) Wash the cells once with ice cold PBS at 300-400 x g and re-suspend in 100-
200µl FACS buffer/PFA fixing buffer.
5) Store at 4°C in darkness and acquire preferably within 24 hours.
2. . Indirect immunostaining of surface antigens
Key Reagents – PBS, staining buffer, FACS buffer, PFA fixing buffer
1) Prepare a single cell suspension using the appropriate protocol and adjust the cell
concentration to 107
cells/ml in stainingbuffer.
2) Dispense 100µl of cell suspension into as many staining tubes as needed
[unstained control, compensation controls, optional isotype and FMO controls, and test
sample].
3) Add the optimized dilution of primary antibodies to the respective tubes and incubate at 4°C
(on ice) for 30 minutes.
4) Wash the cells once with ice cold PBS at 300-400 x g and re-suspend in 100µl staining
buffer.
5) Add the specific secondary antibodies at the proper dilution and incubate the cells at 4°C (on
ice) for 30 minutes in the dark.
6) Wash the cells once with cold PBS at 300-400 x g and re-suspend in 100- 200µl FACS
buffer/PFA fixing buffer.
7) Store at 4°C in darkness and acquire preferably within 24 hours.
3.. General immuno-staining procedure for intracellular antigens 3a.
Permeabilization with methanol
Key Reagents – PBS, staining buffer, FACS buffer, 0.5-4% PFA in PBS [exact concentration of PFA
has to be standardized for every antibody panel], 100% methanol
1) Perform surface staining as per protocols 1 or 2 along with the suitable controls.
2) Aliquot the stained cells in 0.5-4% PFA at 107
cells/ml. Prepare unstained aliquots for the
intracellular staining controls.
3) Fix the cells on ice for 10-30 minutes away from light.
22
6. 8) Add the optimized dilution of primary antibodies to the respective tubes and incubate at 4°C
(on ice) for 30 minutes.
9) Wash the cells once with ice cold PBS at 400-500 x g and re-suspend in 100µl staining
buffer.
10)Add the specific secondary antibodies at the proper dilution and incubate the cells at 4°C (on
ice) for 30 minutes in the dark.
11)Wash the cells once with cold PBS at 400-500 x g and re-suspend in 100- 200µl FACS
buffer/PFA fixing buffer.
12)Store at 4°C in darkness and acquire preferably within 24 hours.
3b. Permeabilization with saponin
Key Reagents – PBS, staining buffer, FACS buffer, 0.5-4% PFA in PBS [exact concentration of PFA
has to be standardized for every antibody panel], 0.1% saponin
1) Perform surface staining as per protocols 1 or 2 along with the suitable controls.
2) Aliquot the stained cells in 0.5-4% PFA at 107
cells/ml. Prepare unstained aliquots for the
intracellular staining controls.
3) Fix the cells on ice for 10-30 minutes away from light.
4) Wash out the fixative at 300-400 x g and add 0.1% saponin.
5) Incubate the cells at room temperature for 15 minutes.
6) Wash out saponin at 300-400 x g and re-suspend the cells in 100µl staining buffer.
7) Dispense 100µl of cell suspension into as many staining tubes as needed
[unstained control, compensation controls, optional isotype and FMO controls, and test
sample].
8) Add the optimized dilution of primary antibodies to the respective tubes and incubate at 4°C
(on ice) for 30 minutes.
9) Wash the cells once with ice cold PBS at 400-500 x g and re-suspend in 100µl staining
buffer.
10)Add the specific secondary antibodies at the proper dilution and incubate the cells at 4°C (on
ice) for 30 minutes in the dark.
11)Wash the cells once with cold PBS at 400-500 x g and re-suspend in 100- 200µl FACS
buffer/PFA fixing buffer.
12)Store at 4°C in darkness and acquire preferably within 24 hours.
4. Intracellular cytokineStimulation and Phospho-immunostaining
23
7. increased production of cytokines inside cells. Refer to the table below as a guideline for common
cell stimulation procedures.
Key Reagents – PBS, staining buffer, FACS buffer, 0.5% PFA in PBS, 0.1% saponin or 100%
methanol, suitable cell stimulant, brefeldin A or monensin
1) Harvest cells using the suitable protocol and aliquot them in tubes at the pre- determined
concentration (depending on cell type and stimulant).
2) Add the specific stimulant and incubate the cells at 37°C for the requisite time. The table
below is a handy reference of different stimulants and incubation time vis-à-vis the target
proteins.
- In the case of staining for secreted cytokines, add brefeldin A or monensin during the
incubation period at the concentration recommended by the manufacturer.
- Set aside some unstimulated aliquots for the unstained control and stained baseline controls.
In Vitro Cell Stimulation Reference Table
24
Target cytokine/phospho
protein
Target cells Stimulant Duration Surface
marker
IL-2 PBMCs PMA (50ng/ml) 4-6 hours CD3
IL-3 T-cells PMA(50ng/ml) + ionomycin
(1µg(ml)
4-6 hours CD4
IL-4 PBMCs PMA(50ng/ml) + ionomycin
(1µg(ml)
4-6 hours CD4
IL-6 PBMCs LPS (100ng/ml) 4-6 hours CD14
IL-10 PBMCs LPS (100ng/ml) 18-24
hours
CD14
GM-CSF
/IFNγ/TNFα/TNFβ
PBMCs PMA(50ng/ml) + ionomycin
(1µg(ml)
4-6 hours CD3
pStat5 PBMCs GM-CSF (20ng/ml) + IL3 (20ng/ml) 15 min. CD123,
CD116
pStat3 PBMCs G-CSF (20ng/ml) + IL6 (20ng/ml) 15 min. CD126,
CD114
pERK PBMCs IL3 (20ng/ml) + IL6 (20ng/ml) +
FLT3L (20ng/ml)
15 min. CD123,
CD126,
CD135
8. 3) Stop the stimulation by fixing the cells with the final concentration of 0.5% PFA.
4) Vortex gently and keep cells on ice for 15 minutes.
5) Wash off the fixative and proceed with surface staining as per protocol1 or 2.
6) Re-suspend cells in the preferred permeabilizing reagent and proceed with the
permeabilization and intracellular staining accordingly as per protocol 3a or 3b (100%
methanol or 0.1% saponin).
5. . Dye efflux staining
Dye exclusion staining is performed to separate live and dead cells, as well as to isolate the rare
stem cell ‘side populations’. If viability staining is included in your regular immunostaining, it should
be performed before any other staining.
5a. Propidium iodide (PI) staining (viability)
Key reagents – PBS, staining buffer, PI solution (10µg/ml in PBS)
1) Harvest the cells and wash once with PBS.
2) Re-suspend cells in staining buffer at 107
cells/ml.
3) Add 5µl of PI stain per 100µl of cell suspension, mix gently and let it stay in the dark for 1
minute.
4) Wash out the dye and re-suspend cells in a suitable volume of staining buffer.
5b. 7-Amino actinomycin D (7-AAD) staining (viability)
Key reagents – PBS, staining buffer, 7-AAD solution (100µg/ml in PBS)
1) Harvest the cells and wash once with PBS.
2) Re-suspend cells in staining buffer at 107
cells/ml.
3) Add 2µl of 7-AAD stain per 100µl cell suspension, mix gently and incubate the cells on ice for
30 minutes.
4) Wash out the dye and re-suspend cells in a suitable volume of staining buffer.
5c. Rhodamine 123 or Hoechst 33342 staining (side population)
Key reagents – PBS, 5% FBS in PBS, staining buffer, Hoechst 33342 solution (1mM in PBS) or
Rho123 solution (10µg/ml in PBS) 25
9. 6. . DNA content and Cell cycle analysis
Key reagents – PBS, staining buffer, PI solution (50µg/ml in PBS), RNAse A (10µg/ml), 70% ethanol
1) Harvest cells and wash once in PBS.
2) Re-suspend cells in staining buffer at 107
cells/ml.
3) Aliquot 500µl of cells into separate tubes (pre-chilled) and add ice cold 70% ethanol dropwise
with gentle vortexing.
4) Keep cells on ice for 1 hour.
5) Wash the cells twice in PBS at 400-500 x g for 10 minutes.
6) Add 1ml of PI solution to the cell pellet and mix well. Add 50µl of RNase to a final
concentration of 0.5µg/ml.
7) Incubate the cells at 4°C overnight.
8) Wash once in PBS and re-suspend in a suitable volume of staining buffer.
Optimization Tips
Sample preparation and cell quality
1) Whenever possible, use freshly isolated cells rather than frozen and thawed cells.
2) To increase viability of thawed cells, perform the initial dilution of the thawed cells at a high
serum concentration (90% FBS in culture medium).
3) When isolating populations rich in adherent cells, use non-tissue culture treated plastic dishes
and tubes.
4) When isolating cells from complex tissues, it is better to perform the steps on ice (except for
the steps involving digestion enzymes) to prevent phagocytosis
and cell lysis.
5) To prepare homogenous single-cell suspensions, gently pipette the cells gently as opposed
to vortexing in order to avoid cell disruption.
6) If the downstream procedure is live cell sorting, it is recommended that cells are counted after
each step to ascertain viability.
7) Minimize dead cells in the final suspension by removing clumps and other debris by sieving
through nylon mesh.
General immuno-staining (fluorescence staining)
26
10. 5) In case an antibody has to be diluted and then stored as aliquots, use the staining buffer for
dilution. Add 0.09% sodium azide to prevent bacterial contamination.
6) Wash the cells once or twice in staining buffer after each incubation step to remove any
unbound antibodies.
7) To amplify the signal for weak antigens, consider using a three step staining
process – antigen binding with biotinylated primary antibody → binding with
streptavidin conjugated secondary antibody → final binding with anti- streptavidin antibody
conjugated to a fluorochrome.
8) Include a viability dye in the antibody cocktail to gate out any dead cells or cell debris.
9) To prevent non-specific Fc receptor staining, add an Fc blocking step or include FBS in the
staining buffer. Alternatively, include an isotype control to subtract any signal contributed by
the Fc receptor staining.
10)It is always better to acquire the cells soon after staining to minimize any fluorochrome
bleaching. In case the cells need to be stored, fix the cells in a
suitable fixative and store at 4°C: for overnight storage 0.5% PFA I a good
choice but for longer durations like several days or even weeks, the recommended fixative
is ethanol.
11)Long term storage in fixative is not recommended as it can significantly increase auto-
fluorescence.
12)Antibody titration is recommended to determine the correct concentration of an antibody for
the optimum signal. Test different dilutions of the antibody to zero in on the lowest
concentration that gives the strongest signal in positive control and the weakest signal in a
negative control.
If the specific antibody concentration of a given unpurified antibody preparation is
unknown, here are our suggested dilutions for various different sources of antibody:
27
Tissue culture
Ascites Whole antiserum Purified antibody
1/100 1/1000 1/500 1 µg/mL
supernatant
Intracellular staining: Fixation and Permeabilization
1) For the staining of secreted proteins like cytokines, a protein transport inhibitor such as
Monensin or Brefeldin A should be added prior to fixation/permeabilization in order to trap
the cytokines inside.
2) For combined surface and intracellular staining, it is advisable to first stain for
the surface markers and then fix/permeabilize as the latter can alter some antigen epitopes
and affect antibody binding.
3) Fixation/permeabilization reagents alter the scatter properties as well as the auto-
fluorescence of cells. Therefore it is recommended to include an unstained control that has
been treated with the same reagents.
11. 4) Binding of antibody to surface antigen can stimulate the cells and alter the expression of
intracellular signaling proteins. Therefore, surface staining should always be performed after
the stimulation.
5) For phosflow staining, the cells should be fixed and permeabilized
immediately after the stimulation as phosphorylation is a transitory phase and quickly pass.
6) Choosing the right permeabilizing agent is extremely important – one can choose between
detergents like saponin or TritonX and methanol.
a. Saponin does not alter the surface antigen epitopes so surface staining can be done
afterwards.
b. TritonX and Tween should be avoided as they can lyse cells if incubated for long.
c. Methanol is compatible with most intracellular antigens and cells treated with
methanol can be stored at -20 to -80°C for an extended
duration without loss of signal.
d. Not all fluorochromes however can withstand methanol treatment. The table below
shows which commonly used dyes are methanol resistant and methanol sensitive.
28
Multicolor panel design
1) Always try to pair the brightest fluorochromes with the weakest expressing antigen. In case
the expression level of an antigen is unknown, it is advisable to use brighter fluorochromes.
2) Avoid spillover or spectral overlap between fluorochromes by spreading them as much as
possible across the spectrum.
3) Avoid fluorochromes that can be excited by more than one laser e.g. APC- Cy7.
4) Always include suitable compensation controls (explained in section 4)
especially if points 3 and 4 above cannot be achieved.
5) It is also advisable to use FMO controls (see section 4) to gate differently stained sub-
populations more accurately.
6) Consider using online multicolor panel designers provided by FACS technology companies.
Methanol Sensitive Methanol Resistant
FITC PE
eFlour 450 PerCP
eFluor 660 APC
Alexa Fluor 647 All tandem dyes
Alexa Fluor 488
12. Example: A suitable 4-color panel to illustrate points 1-3. The color of the boxes correspond to
the lasers that excite the respective fluorochromes.
29
Experimental (Staining) controls
Control What to include Purpose Notes
Unstained control
Unstained cells (incubated in parallel with
your stained samples) that are fully
processed without addition of any
antibodies.
To controlfor
background derived from auto-
fluorescence, and to set the
voltages and negative gates
appropriately.
Used as an
additional negative control.
Comparison to beads can helpto determine the
relative amount of autofluorescence. Try using a
different excitation source if autofluorescence levels
are high.
Isotype
control
Cells incubated with isotype control
antibodies (antibodies usually raised
against an antigen that should not be
present in your cells).
To determine nonspecific
binding of the primary
antibody.
The isotype control should match:
-The host species
-Ig subclass (IgA, IgG, IgD, IgE, or IgM) of the primary or
secondary antibody
-Conjugated to same fluorophore as the
primary
antibody
13. Control What to include Purpose Notes
Internal negative control
Population of cells that do not
express the antigen of interest
and are fully processed.
To avoid false positives
resulting from nonspecific
antibody binding.
Negative control cells are
not always available. Ideally, the fluorescence
intensity of
the internal control should be the same as
the unstained control.
Positive control
Cells known to
express your target of interest.
To avoid false
negatives resulting from a bad
antibody.
Positive control cells are not always available.
Compensation controls
Compensation
controls must match the exact
experimental fluorochrome (with a
similar brightness).
The controls need to be at least as
bright or brighter than any sample
the compensation will be applied
to, and background fluorescence
should be the same for the
positive andnegative controls.
Allows you to remove (compensate
for) spectral overlap.
Spectral overlap should be
compensated for every
fluorophore used.
Single staining in multicolor flow cytometry is
important due to spectral overlap between
different fluorophores. Be sure to collect
enough events tobe statistically significant.
Viability control
Common viability dyes to identify
dead cells include DNA dyes or
protein binding dyes.
Exclusion of dead cells using
viability staining means less non-
specific binding and easier
identification of positively stained
populations.
Using a live/dead stain to remove dead cells
can improve your staining. Dead cells have
greater autofluorescence and increased non-
specific antibody binding, leading to false
positives and reducing the dynamic range.
Fc blocking control
Fc blocking reagents added to
staining procedure; a commercial
Fc block can be used.
To ensure that only antigen specific
binding is observed (Antibody
binding via Fc receptors canlead to
false positives and data that cannot
be interpreted).
Alternatively, the serum of
the host primary antibody can also be used.
For
example, if your antibody is of a mouse isotype,
you could use mouse serum to block non-
specific binding of immunoglobulins/antibodies
to the Fc receptors. For purified PBMCs, 10%
human serum can be used.
30
14. Control What to include Purpose Notes
Fluorescence minus
one (FMO)
controls
The experimental cells stained with all
the fluorophores minus one for each
fluorophore in your multicolor panel.
To detect influence of
fluorescence spread within
detection
channels (especially with brighter
fluorophores).
Important for building multicolor flow
cytometry panels and helps determine where
gates should be set.
Secondary
antibody control
Cells incubated only with the
secondary antibody.
To determine nonspecific binding of the
secondary antibody.
Only necessary if using a secondary
antibody.
31
Troubleshooting Guide
The following guide serves as a checklist for the possible causes and solutions with respect to some of the most
commonly encountered problems from flow cytometry (FACS) experiments. We at Boster Bio are committed to helping
our customers get better results. While the troubleshooting guide below covers a multitude of problems encountered
while performing FACS experiments, we do not expect it to be the exclusive solution to any problems during your
specific experiment. We hope that you will find the information beneficial to you and useful as a reference guide in
troubleshooting any FACS problems you may encounter. If you ever need more assistance with your flow cytometry
experiments, please contact the Boster Support Team by email at support@bosterbio.com
Problem: Weak Fluorescence Intensity or No Fluorescent Signal
Possible Cause Solution
1. The antibodies are degraded or expired
- Ensure that antibodies are stored as per the instructions
of manufacturer.
-Keep track of antibody stocks; make sure products are not expired.
-If titrating antibodies and storing aliquots of the same, add sodium azide in the storage buffer at
0.09%.
2. The fluorescence of the fluorochrome has
faded
- Be sure to store the conjugated antibodies away from
light exposure. Fresh antibodies will be needed.
3. The antibody concentration is too low for detection
- Titrate the antibodies before use to find the optimal
amount to use for your specific experiment.
- Use negative (unstained) and positive controls.
4. Expression of target antigen is too low
- Check literature for antigen expression in different cell
types and use a suitable positive control.
-Whenever possible, use freshly isolated cells as opposed to frozen samples.
-Optimize cell culture/stimulation protocols in case the antigen (e.g. cytokines) expression
depends on prior in vitro treatment.
5. Antigen-antibody binding is sub-optimal
- Check the species specificity of the antibody.
- Optimize the antibody incubation time and temperature.
- Consider using biotinylated primary antibodies and an
15. additional biotin-steptavidin step to amplify the signal.
5. The intracellular antigen is not accessible - Optimize cell permeabilization protocols.
6. The intracellular antigen is getting secreted - Target must be membrane bound or cytoplasmic to be
detected. Try using a Golgi blocker such as Brefeldin A.
7. The surface antigen is getting internalized
- Perform all protocol steps at 4°C and use ice cold
reagents. We recommend you permeablize on ice.
8. The fluorescence on stained cells has bleached
- Acquire the cells immediately after staining.
-Add a fixative (like PFA) to the samples if storing for an extended duration; alcohol fixatives are
best avoided.
9. A low expressing antigen has been paired
with a dim fluorochrome
- Always pair the weak antigens with bright fluorochromes
such as PE or APC.
10. The primary and secondary antibodies are
not compatible
- Use a secondary antibody that was raised against the
species in which the primary antibody was raised.
11. The laser and PMT settings are not compatible with fluorochrome or PMT
voltage is too low for the fluorescent specific channel
- Ensure that proper instrument settings are loaded prior to
acquisition.
-Use suitable positive and negative controls to optimize settings for every fluorochrome.
12. The fluorescent signal is over compensated - Use MFI alignment instead of visual comparison to
compensate.
32
Problem: Excess Fluorescent Signal
Possible Cause Solution
1. The antibody concentration is too high
- Titrate the antibodies before use to find the optimal
amount to use for your specific experiment.
- Use appropriate positive and negative controls.
2. Unbound antibodies are trapped in the cells in
the case of intracellular staining
- Wash the cells adequately after each antibody incubation
step and include Tween or Triton X in wash buffers.
3. A high expressing antigen is paired with
bright fluorochrome
- Always pair strong antigens with dimmer fluorochromes
such as FITC or Pacific Blue.
4. The PMT voltage is too high for the fluorescent specific channel
- Ensure proper instrument settings are loaded prior to
acquisition.
-Use suitable positive and negative controls to optimize settings for every fluorochrome.
5. The fluorescent signal is under-compensated - Use MFI alignment instead of visual comparison to
compensate.
6. Inadequate blocking - Add 1% to 3% blocking agent with antibody as well asa
blocking step (dilute antibodies in blocking solution), and increase the blocking time.
16. Problem: High Background or Non-specific Staining
33
Possible Cause Solution
1. Excess, unbound antibodies are present in
the sample
- Wash cells adequately after every antibody incubation
step.
2. Non-specific cells are targeted
- Include an isotype control to subtract any Fc binding
signal.
-Block the Fc receptors on cells with Fc blockers, BSA or FBS prior to antibody incubation.
- Include additional washing steps.
-Include a secondary antibody-conjugate control to subtract any non-specific signal from the
conjugate.
- Select a secondary antibody that does not cross react.
3. High auto-fluorescence
- Always include an unstained control to subtract the auto-
fluorescence signal.
-For cells with naturally high auto-fluorescence (e.g. neutrophils), use fluorochromes that emit in
the red channel where auto-fluorescence is minimal (e.g. APC).
-If the above solution is not possible, use very bright fluorochromes for these cell types to
amplify the signal above the auto-fluorescence level.
-Avoid storing the cells in a fixative solution for long durations; analyse cells soon after staining
if possible.
4. Presence of dead cells
- Always sieve the cells once before acquiring and sorting
to remove any dead cell debris.
-Include viability dyes like PI or 7-AAD to gate out any dead cells.
-Use freshly isolated cells as opposed to frozen cells whenever possible.
Problem: High Background Scatter or Abnormal Scatter Profile of Cells
Possible Cause Solution
1. The cells are lysed or damaged
- Optimize sample preparation to avoid cell lysis.
-If possible, do not vortex or centrifuge cells at high speeds.
- Use fresh buffers.
- Avoid storing the stained cells for long durations.
2. Bacterial contamination
- Store stained cells and antibodies properly to avoid
bacterial growth. Bacteria will exhibit low levels of auto fluorescence. Practice proper sterile cell
culture techniques to prevent bacterial contamination.
17. 3. Incorrect instrument settings for scatter
- Ensure proper instrument settings are loaded prior to
acquisition.
-Use fresh, healthy cells to correctly set the FSC and SSC settings for that cell type.
4. Presence of dead cells
- Always sieve the cells once before acquiring and sorting
to remove any dead cell debris.
-Use freshly isolated cells as opposed to frozen cells whenever possible.
5. Presence of un-lysed RBCs (red blood cells)
- Ensure RBC cells lysis is complete – check using a
microscope.
- Use fresh RBC lysis buffer.
- Wash as many times as needed to remove RBC debris.
-If using PBMCs after a Ficoll gradient, optimize the procedure to minimize RBC
contamination in the lymphocyte interphase.
34
Problem: Abnormal Event Rate
Possible Cause Solution
1. Event rate is low due to low cell number
- Keep the minimum cell count at 1X106
/ml.
- Ensure the cells are mixed well with gentle pipetting.
2. Event rate is low due to sample clumping
- Always sieve the cells once prior to acquiring and sorting
to remove debris.
-Ensure the cells are mixed well with gentle pipetting before staining, and again before running
your samples.
3. Incorrect instrument settings - Adjust the threshold parameter as needed.
4. No events due to a clogged sample injection tube - Unclog flow cytometer injection tube as per instrument
manufacturer's instructions (typically run 10% bleach for5- 10 min, followed by dH2O for 5-10 min).
5. Event rate is too high due to concentrated
sample
- Dilute the cell count to 1X106
/ml.
6. Event rate is too high due to air in flow cells
and/or sheath filter
- Refer to the instrument manual for the appropriate steps.
Problem: Loss of Epitope
Possible Cause Solution
1. Excessive paraformaldehyde
- Paraformaldehye can release methanol in its breakdown,
which may affect the staining. Make sure to use only1% paraformaldehyde.
2. Sample was not kept on ice - Keep antibodies at 4°C to prevent loss of activity. This