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
Organ culture involves maintaining small fragments of whole organs or tissues in culture media while retaining their three-dimensional structure and spatial distribution of cells. There are several methods of organ culture including culturing on plasma clots, agar, liquid media, or raft methods. Organ culture has various applications and allows studying cell interactions in a way that mimics the in vivo organ. It is currently being used to develop replacement organs and tissues for applications such as growing bladders, lungs, and heart patches. While progress is being made, developing fully functional human organs remains a challenge.
This document discusses stem cell culture and provides definitions, classifications, and methods for culturing different types of stem cells. It summarizes the history of stem cell research from 1981 to present. It describes embryonic stem cells, adult stem cells including bone marrow and umbilical cord stem cells. Methods are outlined for isolating and culturing stem cells from bone marrow and umbilical cord. Advantages and disadvantages of different stem cell sources are compared.
Introduction
Primary Culture
Steps In Primary Culture
Isolation Of Tissue
Dissection And/Or Disaggregation
Types Of Primary Culture
Primary Explant Culture
Enzymatic Disaggregation
Mechanical Disaggregation
Cell Line( Finite & Continuous)
Naming A Cell Line
Choosing A Cell Line
Maintenance Of Cell Line
Conclusion
reference
Bioreactors for animal cell suspension cultureGrace Felciya
This document discusses bioreactors for animal cell suspension culture. It begins by introducing animal cell culture and some key developments that enabled it. There are two main types of culture: primary culture using explants or enzymes, and secondary culture which is derived from primary culture. Cells can be anchorage-dependent, growing in monolayers, or non-anchorage dependent, growing in suspension. Bioreactors provide conditions for mass cultivation of suspension cells. Properties of animal cells require gentle mixing and aeration in bioreactors. Common bioreactor types for suspension culture include stirred tank, continuous flow, and airlift fermentors. Perfusion culture allows continuous medium exchange to achieve high cell densities and productivity.
- Cell culture media is the most important factor in cell culture technology as it supports cell survival, proliferation, and functions. This directly impacts research results and biopharmaceutical production.
- There are two main types of cell culture media: natural media consisting of biological fluids/extracts, and artificial/synthetic media composed of defined components like salts, nutrients, and serum.
- Early natural media included blood plasma and tissue extracts. Later, defined artificial media were developed with basal solutions and supplements to meet specific cell needs. Serum remains a common artificial media supplement providing growth factors and nutrients.
Cell culture media are designed to support the growth of cells outside their natural environment. They generally contain amino acids, salts, glucose, vitamins and other nutrients. Media can be natural (containing biological fluids) or artificial/synthetic. Artificial media are grouped into serum-containing, serum-free, chemically defined, and protein-free categories based on their ingredients. Key components of media include buffers, amino acids like glutamine, vitamins, inorganic salts, carbohydrates, proteins, lipids, trace elements, and supplements specific to cell lines. Selection of the appropriate medium depends on the cell type and purpose of culture. Primary cells especially benefit from ready-to-use conditioned media.
Stem cells have the ability to differentiate into various cell types and can help treat many medical conditions. There are two main types - embryonic stem cells which are pluripotent and can form nearly every cell type, and adult stem cells which are multipotent and usually form a limited number of cell types. Recent research has shown that mature cells can be reprogrammed into pluripotent stem cells through nuclear transfer or the introduction of specific factors. This opens up new possibilities for regenerative medicine and treating diseases.
This document discusses cell culture contamination, including sources, types, signs, and methods for monitoring and preventing contamination. Common sources of contamination include failed sterilization of equipment, airborne particulates, and poorly maintained incubators. Major types of microbial contaminants are bacteria, molds, mycoplasma, yeasts, and viruses. Signs of contamination vary but may include cloudy cultures, pH changes, and visible microbes under a microscope. Proper aseptic technique and regular monitoring of cell cultures can help reduce contamination. Cross-contamination between cell lines must also be avoided.
Organ culture involves maintaining small fragments of whole organs or tissues in culture media while retaining their three-dimensional structure and spatial distribution of cells. There are several methods of organ culture including culturing on plasma clots, agar, liquid media, or raft methods. Organ culture has various applications and allows studying cell interactions in a way that mimics the in vivo organ. It is currently being used to develop replacement organs and tissues for applications such as growing bladders, lungs, and heart patches. While progress is being made, developing fully functional human organs remains a challenge.
This document discusses stem cell culture and provides definitions, classifications, and methods for culturing different types of stem cells. It summarizes the history of stem cell research from 1981 to present. It describes embryonic stem cells, adult stem cells including bone marrow and umbilical cord stem cells. Methods are outlined for isolating and culturing stem cells from bone marrow and umbilical cord. Advantages and disadvantages of different stem cell sources are compared.
Introduction
Primary Culture
Steps In Primary Culture
Isolation Of Tissue
Dissection And/Or Disaggregation
Types Of Primary Culture
Primary Explant Culture
Enzymatic Disaggregation
Mechanical Disaggregation
Cell Line( Finite & Continuous)
Naming A Cell Line
Choosing A Cell Line
Maintenance Of Cell Line
Conclusion
reference
Bioreactors for animal cell suspension cultureGrace Felciya
This document discusses bioreactors for animal cell suspension culture. It begins by introducing animal cell culture and some key developments that enabled it. There are two main types of culture: primary culture using explants or enzymes, and secondary culture which is derived from primary culture. Cells can be anchorage-dependent, growing in monolayers, or non-anchorage dependent, growing in suspension. Bioreactors provide conditions for mass cultivation of suspension cells. Properties of animal cells require gentle mixing and aeration in bioreactors. Common bioreactor types for suspension culture include stirred tank, continuous flow, and airlift fermentors. Perfusion culture allows continuous medium exchange to achieve high cell densities and productivity.
- Cell culture media is the most important factor in cell culture technology as it supports cell survival, proliferation, and functions. This directly impacts research results and biopharmaceutical production.
- There are two main types of cell culture media: natural media consisting of biological fluids/extracts, and artificial/synthetic media composed of defined components like salts, nutrients, and serum.
- Early natural media included blood plasma and tissue extracts. Later, defined artificial media were developed with basal solutions and supplements to meet specific cell needs. Serum remains a common artificial media supplement providing growth factors and nutrients.
Cell culture media are designed to support the growth of cells outside their natural environment. They generally contain amino acids, salts, glucose, vitamins and other nutrients. Media can be natural (containing biological fluids) or artificial/synthetic. Artificial media are grouped into serum-containing, serum-free, chemically defined, and protein-free categories based on their ingredients. Key components of media include buffers, amino acids like glutamine, vitamins, inorganic salts, carbohydrates, proteins, lipids, trace elements, and supplements specific to cell lines. Selection of the appropriate medium depends on the cell type and purpose of culture. Primary cells especially benefit from ready-to-use conditioned media.
Stem cells have the ability to differentiate into various cell types and can help treat many medical conditions. There are two main types - embryonic stem cells which are pluripotent and can form nearly every cell type, and adult stem cells which are multipotent and usually form a limited number of cell types. Recent research has shown that mature cells can be reprogrammed into pluripotent stem cells through nuclear transfer or the introduction of specific factors. This opens up new possibilities for regenerative medicine and treating diseases.
This document discusses cell culture contamination, including sources, types, signs, and methods for monitoring and preventing contamination. Common sources of contamination include failed sterilization of equipment, airborne particulates, and poorly maintained incubators. Major types of microbial contaminants are bacteria, molds, mycoplasma, yeasts, and viruses. Signs of contamination vary but may include cloudy cultures, pH changes, and visible microbes under a microscope. Proper aseptic technique and regular monitoring of cell cultures can help reduce contamination. Cross-contamination between cell lines must also be avoided.
This document discusses different substrates used for animal cell culture, including glass, plastics, and metals. It also describes two types of cell suspension culture: batch culture and continuous culture. Batch culture involves growing cells in a fixed volume of medium until growth limiting factors are reached. Continuous culture maintains constant culture conditions through continuous addition of fresh medium and removal of used medium and cells. The chemostat and turbidostat are two common systems used for continuous culture that maintain steady state growth. Cell suspension culture is important for studies of cell physiology, biochemistry, and production of secondary metabolites.
Cell viability and proliferation assays measure aspects of cellular health and function, such as membrane integrity, metabolic activity, and DNA synthesis. Common assays include MTT, which measures mitochondrial activity; ATP assays, which measure ATP concentration as a marker of viability; Sulforhodamine B, which binds cellular proteins to measure biomass; and propidium iodide staining, which detects compromised membranes. These assays are useful for screening drug toxicity and effects on cell growth.
This document discusses different types of cell and organ culture. It describes four main types of culture media: natural media which are obtained from natural sources but have unknown compositions; and three types of artificial media - serum containing media which typically contain 2-10% fetal bovine serum, serum-free defined media which have consistent compositions and reduce contamination risk, and protein-free and chemically defined media. It also outlines techniques for primary cell culture, establishing cell lines, and organ culture methods like plasma clot, agar gel, and raft methods.
This document provides a history and overview of animal cell culture techniques. It discusses the development of cell culture media and reagents used to support cell growth in vitro. It also describes different techniques for culturing mammalian cells, tissues, and organs, including organ culture, explant culture, and cell culture. The goal of animal cell culture is to maintain cells, tissues, or organs outside of their natural environment for research purposes.
This document describes different types of cell cultures. There are three main types: primary cell culture, secondary cell culture, and cell lines. Primary cell culture involves directly separating cells from parent tissue through enzymatic or mechanical means and maintaining their growth in culture medium. Secondary cell culture occurs when a primary culture is sub-cultured and becomes known as a cell line. Cell lines can be finite, with limited life spans, or continuous, which are immortal in culture. Continuous cell lines show properties like absence of contact inhibition and anchorage dependence.
Biology and characterization of the cell cultureKAUSHAL SAHU
This document provides an overview of cell culture techniques. It discusses the history of cell culture beginning in the late 1800s. Key terms like cell line, normal cell line, and continuous cell line are defined. The document outlines the biology of cultured cells including adhesion, proliferation, differentiation, and energy metabolism. It also discusses the origin of cultured cells and characterization techniques for authenticating cell lines such as morphology, chromosome content, and DNA analysis. The applications of animal cell culture and conclusions are presented.
This document discusses 3D cell culture techniques. It defines 3D cell culture as permitting biological cells to grow and interact in all three dimensions, mimicking their natural environment. This is an improvement over 2D cultures where cells grow unnaturally. The document describes various 3D culture methods including scaffold-based techniques using polymeric scaffolds or biological scaffolds, and non-scaffold methods like hanging drop plates, spheroid plates, microfluidics, and gels. It also discusses bioreactors and lists applications of 3D cultures.
Histotypic culture involves growing cell lines in a three-dimensional matrix at high density to form tissue-like structures. Common techniques include using gels, sponges, hollow fibers, spheroids, and rotating chambers to provide a 3D environment that allows cells to organize similarly to how they would in tissues. This allows for the study of processes like drug penetration and cell differentiation that are not possible with traditional 2D cultures. While histotypic cultures provide a model for certain tissue functions, they also face challenges like loss of cell differentiation over time.
Primary and established cell line cultureKAUSHAL SAHU
Introduction
Primary Culture
Steps of Primary Culture
Isolation Of Tissue
Dissection And Disaggregation
Types Of Primary Culture
Primary Explants Culture
Enzymatic Disaggregation
Mechanical Disaggregation
Cell Line( Finite & Continuous)
Naming A Cell Line
Choosing A Cell Line
Maintenance Of Cell Line
Conclusion
Reference
INTRODUCTION
HISTORY
NEED OF SYNCHRONIZATION
SYNCHRONOUS CULTURES CAN BE OBTAINED IN SEVERAL WAYS:
Physical fractionation .
Chemical appro ach
CENTRIFUGAL ELUTRIATION
Inhibition of DNA synthesis
Nutritional deprivation
SYNCHRONIZATION AT LOW TEMPERATURE
CELLULAR TOTIPOTENCY
SOME HIGHLIGHTS OF CELL SYNCHRONIZATION
REFERENCES
8. Biology and characterization of cultured cellsShailendra shera
Immediate environment and environment of surrounding medium governs the various properties of cell. The in vitro condition markedly affects the cellular property of cultured cells. For e.g. Reduction in Cell–cell and cell-material interaction. Therefore, it is imperative to develop understanding of biology of cells in response to various environmental conditions. Characterization of cells helps to identify the origin, purity and authenticity of cells and cell lines.
History of animal tissue culture and natural surroundings for animal cellNeeraj Chauhan
This document discusses the history of animal tissue culture and factors that affect culturing animal cells. It notes that Roux in 1885 was the first to culture embryonic chick cells and Harrison in 1907 successfully cultured nerve cells. Key events over 130 years included development of defined media and serum-free media. Factors that impact cell culture choice include cell yield, whether cells are monolayer or suspension, venting, sampling needs, growth uniformity, and cost. Environmental factors like pH, temperature, gas phase, osmolarity, foaming, and viscosity must also be controlled to maintain optimal cell growth conditions.
Organ culture technique in synthetic media- animal tissue culture neeru02
Organ culture is a development from tissue culture that allows for the culture of pieces of organs on artificial media to accurately model organ functions in various states. Special culture methods are required as organs require high oxygen levels. Organ pieces can be cultured on plasma clots, agar, raft methods using lens paper or rayon, grid methods, or in liquid media using supports like gauze or rafts. Organ culture faces limitations as results may not match whole animal studies due to lack of in vivo drug metabolism.
This document discusses different types of cell culture, including primary culture, secondary culture, cell lines, and established cell line culture. It describes the process of isolating and culturing cells from tissue samples, including enzymatic and mechanical disaggregation of tissues. The importance of physical environment and culture media for growing cells in vitro is highlighted. Different types of culture media like serum-containing, serum-free, and chemically defined media are also summarized.
Immunoelectrophoresis is a technique that combines electrophoresis and immunodiffusion to separate and identify antigen components in a mixture. It involves applying an electric current to separate antigens in agar gel wells, then allowing the antigens to diffuse and react with antibodies placed in troughs cut into the gel. This results in the formation of precipitin lines that indicate reactions between individual antigens and antibodies, allowing different antigens to be identified based on the lines' position and shape. Immunoelectrophoresis is used in medical diagnostics to detect abnormal proteins and monitor antigen purity.
As opposed to common belief, the measurement of growth in cell culture is fairly simple. Most of the tecchniques that are applied for measurement of microbial growth can be applied to cell culture.Of course with some modification. This presentation exactly explains growth measurement techniques with respect to cell culture. At the end you will also find sample multiple choice questions for practice.
The document discusses embryonic development from an embryologist's perspective. It covers the overall view of embryonic development from conception through harvest and techniques. The source and significance of embryonic development is also examined at a high level.
Introduction
What is cloning?
Why we want to do cloning?
History
Technique of cell cloning
Dolly – the sheep
Species cloned
Why persue animal cloning research?
Conclusion
Introduction
What is cloning?
Why we want to do cloning?
History
Technique of cell cloning
Dolly – the sheep
Species cloned
Why persue animal cloning research?
Conclusion
This document discusses cell separation methods used in downstream processing. It describes filtration and centrifugation as the two main methods. For filtration, it explains the theory and types of filters used like plate and frame, pressure leaf, and rotary vacuum filters. For centrifugation, it outlines stock's law and describes basket, tubular bowl, solid bowl (decanter), multi-chamber, and disc bowl centrifuges. Cross flow filtration is also introduced as an alternative to overcome issues with other filtration methods. In under 3 sentences, this document provides an overview of cell separation techniques used in downstream processing and discusses filtration and centrifugation methods in detail.
This work is done in IIT-M (Indian Institute of Technology- Madras) with help of Indian Academy of Science during June 2011-Oct 2011 under Dr Karunagaran Devarajan sir
This document discusses different substrates used for animal cell culture, including glass, plastics, and metals. It also describes two types of cell suspension culture: batch culture and continuous culture. Batch culture involves growing cells in a fixed volume of medium until growth limiting factors are reached. Continuous culture maintains constant culture conditions through continuous addition of fresh medium and removal of used medium and cells. The chemostat and turbidostat are two common systems used for continuous culture that maintain steady state growth. Cell suspension culture is important for studies of cell physiology, biochemistry, and production of secondary metabolites.
Cell viability and proliferation assays measure aspects of cellular health and function, such as membrane integrity, metabolic activity, and DNA synthesis. Common assays include MTT, which measures mitochondrial activity; ATP assays, which measure ATP concentration as a marker of viability; Sulforhodamine B, which binds cellular proteins to measure biomass; and propidium iodide staining, which detects compromised membranes. These assays are useful for screening drug toxicity and effects on cell growth.
This document discusses different types of cell and organ culture. It describes four main types of culture media: natural media which are obtained from natural sources but have unknown compositions; and three types of artificial media - serum containing media which typically contain 2-10% fetal bovine serum, serum-free defined media which have consistent compositions and reduce contamination risk, and protein-free and chemically defined media. It also outlines techniques for primary cell culture, establishing cell lines, and organ culture methods like plasma clot, agar gel, and raft methods.
This document provides a history and overview of animal cell culture techniques. It discusses the development of cell culture media and reagents used to support cell growth in vitro. It also describes different techniques for culturing mammalian cells, tissues, and organs, including organ culture, explant culture, and cell culture. The goal of animal cell culture is to maintain cells, tissues, or organs outside of their natural environment for research purposes.
This document describes different types of cell cultures. There are three main types: primary cell culture, secondary cell culture, and cell lines. Primary cell culture involves directly separating cells from parent tissue through enzymatic or mechanical means and maintaining their growth in culture medium. Secondary cell culture occurs when a primary culture is sub-cultured and becomes known as a cell line. Cell lines can be finite, with limited life spans, or continuous, which are immortal in culture. Continuous cell lines show properties like absence of contact inhibition and anchorage dependence.
Biology and characterization of the cell cultureKAUSHAL SAHU
This document provides an overview of cell culture techniques. It discusses the history of cell culture beginning in the late 1800s. Key terms like cell line, normal cell line, and continuous cell line are defined. The document outlines the biology of cultured cells including adhesion, proliferation, differentiation, and energy metabolism. It also discusses the origin of cultured cells and characterization techniques for authenticating cell lines such as morphology, chromosome content, and DNA analysis. The applications of animal cell culture and conclusions are presented.
This document discusses 3D cell culture techniques. It defines 3D cell culture as permitting biological cells to grow and interact in all three dimensions, mimicking their natural environment. This is an improvement over 2D cultures where cells grow unnaturally. The document describes various 3D culture methods including scaffold-based techniques using polymeric scaffolds or biological scaffolds, and non-scaffold methods like hanging drop plates, spheroid plates, microfluidics, and gels. It also discusses bioreactors and lists applications of 3D cultures.
Histotypic culture involves growing cell lines in a three-dimensional matrix at high density to form tissue-like structures. Common techniques include using gels, sponges, hollow fibers, spheroids, and rotating chambers to provide a 3D environment that allows cells to organize similarly to how they would in tissues. This allows for the study of processes like drug penetration and cell differentiation that are not possible with traditional 2D cultures. While histotypic cultures provide a model for certain tissue functions, they also face challenges like loss of cell differentiation over time.
Primary and established cell line cultureKAUSHAL SAHU
Introduction
Primary Culture
Steps of Primary Culture
Isolation Of Tissue
Dissection And Disaggregation
Types Of Primary Culture
Primary Explants Culture
Enzymatic Disaggregation
Mechanical Disaggregation
Cell Line( Finite & Continuous)
Naming A Cell Line
Choosing A Cell Line
Maintenance Of Cell Line
Conclusion
Reference
INTRODUCTION
HISTORY
NEED OF SYNCHRONIZATION
SYNCHRONOUS CULTURES CAN BE OBTAINED IN SEVERAL WAYS:
Physical fractionation .
Chemical appro ach
CENTRIFUGAL ELUTRIATION
Inhibition of DNA synthesis
Nutritional deprivation
SYNCHRONIZATION AT LOW TEMPERATURE
CELLULAR TOTIPOTENCY
SOME HIGHLIGHTS OF CELL SYNCHRONIZATION
REFERENCES
8. Biology and characterization of cultured cellsShailendra shera
Immediate environment and environment of surrounding medium governs the various properties of cell. The in vitro condition markedly affects the cellular property of cultured cells. For e.g. Reduction in Cell–cell and cell-material interaction. Therefore, it is imperative to develop understanding of biology of cells in response to various environmental conditions. Characterization of cells helps to identify the origin, purity and authenticity of cells and cell lines.
History of animal tissue culture and natural surroundings for animal cellNeeraj Chauhan
This document discusses the history of animal tissue culture and factors that affect culturing animal cells. It notes that Roux in 1885 was the first to culture embryonic chick cells and Harrison in 1907 successfully cultured nerve cells. Key events over 130 years included development of defined media and serum-free media. Factors that impact cell culture choice include cell yield, whether cells are monolayer or suspension, venting, sampling needs, growth uniformity, and cost. Environmental factors like pH, temperature, gas phase, osmolarity, foaming, and viscosity must also be controlled to maintain optimal cell growth conditions.
Organ culture technique in synthetic media- animal tissue culture neeru02
Organ culture is a development from tissue culture that allows for the culture of pieces of organs on artificial media to accurately model organ functions in various states. Special culture methods are required as organs require high oxygen levels. Organ pieces can be cultured on plasma clots, agar, raft methods using lens paper or rayon, grid methods, or in liquid media using supports like gauze or rafts. Organ culture faces limitations as results may not match whole animal studies due to lack of in vivo drug metabolism.
This document discusses different types of cell culture, including primary culture, secondary culture, cell lines, and established cell line culture. It describes the process of isolating and culturing cells from tissue samples, including enzymatic and mechanical disaggregation of tissues. The importance of physical environment and culture media for growing cells in vitro is highlighted. Different types of culture media like serum-containing, serum-free, and chemically defined media are also summarized.
Immunoelectrophoresis is a technique that combines electrophoresis and immunodiffusion to separate and identify antigen components in a mixture. It involves applying an electric current to separate antigens in agar gel wells, then allowing the antigens to diffuse and react with antibodies placed in troughs cut into the gel. This results in the formation of precipitin lines that indicate reactions between individual antigens and antibodies, allowing different antigens to be identified based on the lines' position and shape. Immunoelectrophoresis is used in medical diagnostics to detect abnormal proteins and monitor antigen purity.
As opposed to common belief, the measurement of growth in cell culture is fairly simple. Most of the tecchniques that are applied for measurement of microbial growth can be applied to cell culture.Of course with some modification. This presentation exactly explains growth measurement techniques with respect to cell culture. At the end you will also find sample multiple choice questions for practice.
The document discusses embryonic development from an embryologist's perspective. It covers the overall view of embryonic development from conception through harvest and techniques. The source and significance of embryonic development is also examined at a high level.
Introduction
What is cloning?
Why we want to do cloning?
History
Technique of cell cloning
Dolly – the sheep
Species cloned
Why persue animal cloning research?
Conclusion
Introduction
What is cloning?
Why we want to do cloning?
History
Technique of cell cloning
Dolly – the sheep
Species cloned
Why persue animal cloning research?
Conclusion
This document discusses cell separation methods used in downstream processing. It describes filtration and centrifugation as the two main methods. For filtration, it explains the theory and types of filters used like plate and frame, pressure leaf, and rotary vacuum filters. For centrifugation, it outlines stock's law and describes basket, tubular bowl, solid bowl (decanter), multi-chamber, and disc bowl centrifuges. Cross flow filtration is also introduced as an alternative to overcome issues with other filtration methods. In under 3 sentences, this document provides an overview of cell separation techniques used in downstream processing and discusses filtration and centrifugation methods in detail.
This work is done in IIT-M (Indian Institute of Technology- Madras) with help of Indian Academy of Science during June 2011-Oct 2011 under Dr Karunagaran Devarajan sir
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.
This document describes the methods for conducting an in vitro chromosomal aberration test. White blood cells are cultured from human donors and exposed to test substances like cyclophosphamide. Cells are harvested at different timepoints throughout the cell cycle. Slides are prepared, stained, and analyzed under a microscope to identify chromosomal abnormalities like deletions, duplications, inversions, and translocations. Acceptance criteria include having homogenous results between replicates, aberration frequencies within historical controls, and a concentration-dependent response to a test substance. A conclusion on the potential of a substance to induce chromosomal aberrations is based on statistical analyses.
Flow cytometry uses laser-based technology to identify and quantify cell populations in a fluid suspension. Cells flow through the system and are interrogated by detectors that measure their physical properties like size and complexity. It can identify normal vs abnormal cells, differentiate cell types, and quantify tumor infiltration. Key components include lasers, fluidics to align cells, optical systems to filter light signals, and detectors to convert fluorescence into electrical signals for analysis. It allows multiparameter analysis using antibody panels to detect cell surface and intracellular antigens, aiding diagnosis and monitoring of hematological malignancies.
1) The document summarizes different types of cell viability assays, including dye exclusion assays, colorimetric assays, fluorometric assays, and luminometric assays.
2) Specific assays discussed in more detail include trypan blue exclusion assay, MTT assay, SRB assay, and 5-CFDA-AM fluorometric assay.
3) The assays are described in terms of their principles, protocols, calculation methods, and applications in measuring cell viability and screening drug responses.
This document discusses various techniques for enumerating microorganisms, including direct and indirect methods. Direct methods involve directly counting microbes under a microscope, such as using a counting chamber (e.g. Petri-Hausser chamber) for direct microscopic count. Indirect methods estimate the number of microbes using other indicators, like standard plate count which counts colonies grown from diluted samples, membrane filtration which filters microbes for colony counting, most probable number which estimates concentrations through liquid broth growth at serial dilutions, turbidity testing using spectrophotometers, and measuring metabolic activity or dry weight.
Anticancer activity of six selected natural c ompounds of some camroonian med...Jitendra Shandilya
This document summarizes a study that investigated the cytotoxicity of six natural compounds against various cancer cell lines. The study found that compounds 1 and 2 were able to reduce the proliferation of 3 cell lines by up to 50% at 20 micrograms/mL. Compounds 1 and 2 showed IC50 values below 20 micrograms/mL against 12 of 14 and 14 of 14 cell lines, respectively. Compound 1 induced caspase 3/7 activity, suggesting it induces apoptosis. Both compounds inhibited blood vessel growth in quail eggs by around 60%, indicating their potential to inhibit tumor promotion in vivo. The study confirms the cytotoxic potential of these compounds, particularly compounds 1 and 2.
Stem cell enumeration involves quantifying stem cells, typically through CD34+ cell counting. Key methods include mononuclear cell counts, colony forming unit assays, and CD34+ enumeration via flow cytometry. For flow cytometry, the ISHAGE protocol is commonly used and involves sequential gating of lymphocytes, CD34+ cells, and viable cells. Accurate stem cell quantification is important for determining transplant timing and adequacy.
Examples of Quantification Techniques- Hemocytometry, UV-Vis Spectrophotometr...Jacob Feste
This document describes an experiment that performed spectrophotometry on citrate-gold nanoparticles (C-AuNPs) of varying sizes to analyze how size influences optical properties. C-AuNPs with diameters of 5, 10, 30, and 60 nm were serially diluted and measured. Peak wavelengths of 520 nm, 530 nm, 620 nm, and 630 nm were observed, suggesting these correspond to particle sizes from smallest to largest. The extinction coefficient of samples containing estimated 10 nm particles was calculated as 0.2152 M-1cm-1 from absorbance versus concentration data. The results demonstrate how size affects the optical properties of nanoparticles.
The hemocytometer is a specialized microscope slide with an etched grid used for manually counting cells in blood specimens or cell cultures. It has a precise counting chamber that allows accurate counting of cells suspended in a known small volume of liquid. To use it, a cell sample is loaded into the chamber, cells in sections of the grid are counted under a microscope, and the cell concentration per volume unit is calculated based on the counts, grid area, and loaded volume.
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.
The document describes the direct microscopic method for enumerating bacterial cells using a Petroff-Hausser counting chamber. The counting chamber contains squares that delimit a known sample volume. Cells are counted within the squares under a microscope and the total number of cells in the original sample is extrapolated based on the number counted and sample dilutions. Living and dead cells can be distinguished using a dye that is only permeable to dead cells.
This document describes several methods for enumerating or counting bacteria in a sample, including viable plate count, direct microscopic count, and turbidity count. The viable plate count method involves making serial dilutions of a sample and counting the number of colonies that grow on an agar plate, then multiplying by the dilution factor to determine the concentration in the original sample. The direct microscopic count uses a counting chamber to directly view and count bacteria under a microscope. The turbidity count uses spectrophotometry to measure the turbidity or cloudiness of diluted samples, which correlates to the number of bacteria present based on a generated standard curve. Procedures for each method are provided.
Laser scanning cytometry and liquid based cytologyanaonline
Liquid based cytology (LBC) has been introduced to improve cervical screening. It uses a liquid preservative instead of smearing cells directly on a slide. This allows automated processing to disperse cells and transfer them evenly to a slide. LBC reduces sampling errors, improves cell preservation and slide quality compared to conventional smears. However, it requires specialized equipment and is more expensive. Laser scanning cytometry is a technique that can analyze individual cells from LBC samples. It provides quantitative measurements and complements flow cytometry by allowing analysis of adherent cells and solid tissues.
Laser scanning cytometry and liquid based cytologyanaonline
Liquid based cytology (LBC) has been introduced to improve cervical screening by limiting some of the limitations of conventional pap smears. With LBC, a sample is collected in a preservative solution rather than directly smeared onto a slide. This liquid sample is then processed using an automated system that disperses cells, collects them on a filter, and transfers them onto a slide. LBC results in slides with cleaner backgrounds and better cell dispersion, making screening easier and more accurate. However, LBC equipment and solutions can be more expensive compared to conventional pap smears. Laser scanning cytometry is a technique that can be used to analyze cells collected with LBC.
The MTT assay and the MTS assay are colorimetric assays for measuring the activity of enzymes that reduce MTT or close dyes (XTT, MTS, WSTs) to formazan dyes, giving a purple color The main application allows to assess the viability (cell counting) and the proliferation of cells (cell culture assays)
It can also be used to determine cytotoxicity of potential medicinal agents and toxic materials, since those agents would stimulate or inhibit cell viability and growth
This document discusses polymerase chain reaction (PCR) and real-time PCR techniques. It begins with an overview of using PCR to study gene expression through RNA extraction, cDNA synthesis, and either end point PCR or real-time PCR. Real-time PCR allows for simultaneous amplification and quantification of specific nucleic acid sequences. It describes the basic components and steps of real-time PCR, including different chemistries used and quantification methods. The document emphasizes the importance of controls and melt curve analysis to validate real-time PCR results.
Electrophoresis of LDH Isoenzymes and Activity StainingASHIKH SEETHY
The slides prepared for MD(Biochemistry) and MSc (Biochemistry) teaching comprehensively covers isoenzymes, isoforms, clinical utility of Lactate Dehydrogenase (LDH), LDH isoenzymes and basics of zymography.
Download and view in presenter mode for better visual experience.
Comprehensive description of various primary dyslipidemias, cholesterol transport and molecular mechanisms involved.
View in slideshow after downloading for better experience.
Prepared in Dec 2013.
Use slideshow after downloading for better viewing. The slides cover altered metabolism in cancer with a focus on Warburg effect and drug targeting of metabolic pathways for cancer treatment.
Prepared in Oct 2014
Intracellular Traffic and Sorting of ProteinsASHIKH SEETHY
Describes intra-cellular trafficking of proteins, protein sorting, clinical aspects of protein targeting, and vesicle transport.
Download and view in slide show mode for better viewing.
Slides prepared MBBS Biochemistry lectures. Includes description of hormone signaling, hormone actions, detailed description of insulin and diabetes mellitus, metabolic syndrome, thyroid hormones, calcium and phosphate homeostasis, vitamin D and PTH.
Prepared in Nov 2015
Introduction to CRISPR Cas9 technology. View in slide show after downloading for better viewing. Description is minimal, but it will be worth going through the slides that are full of pictures, if you have a minimal understanding of CRISPR.
Prepared in Oct 2015
The presentation outlines aspects of immunity against cancer, evasion strategies by cells, immunotherapy in cancer, cancer vaccines etc. Download and view the slideshow for better experience.
Prepared in Sept 2014
Serum Protein and Albumin-Globulin RatioASHIKH SEETHY
For MBBS Biochemistry Practical. Explains various methods of protein estimation and estimation of AG ratio, conditions leading to alterations in AG ratio etc.
The slides explain history of Prion diseases, proposed mechanisms of pathogenesis, investigations and proposed treatment options. Pl watch after downloading as the slides are mostly animated.
Prepared in June 2014
A comprehensive coverage of Enzymes including basics, mechanisms of enzyme catalysis, enzyme inhibition and clinical applications, mostly based on Stryer- Biochemistry. The slides were intended for MBBS teaching, but should benefit the students of Biochemistry and allied sciences.
Prepared in Sept 2015
This document discusses sodium and potassium levels in the body. It begins by outlining the distribution of sodium, potassium, and water in the body and their roles in homeostasis. It then describes pathological conditions that can result from imbalances in sodium and potassium levels. Various techniques for estimating serum sodium and potassium are also outlined, including ion selective electrodes, atomic absorption spectroscopy, and flame emission photometry.
Estimation of Serum Cholesterol and HDLASHIKH SEETHY
This document provides information on estimating serum cholesterol and HDL levels:
- It describes the objectives, functions of cholesterol, cholesterol synthesis, lipoproteins, causes of hyperlipidemia, desirable cholesterol levels, laboratory estimation methods for total cholesterol and HDL cholesterol, and protocols for performing the estimations.
- A case study example is provided at the end to estimate total cholesterol, HDL cholesterol, and calculate LDL cholesterol for a patient with a family history of early myocardial infarction.
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.
When I was asked to give a companion lecture in support of ‘The Philosophy of Science’ (https://shorturl.at/4pUXz) I decided not to walk through the detail of the many methodologies in order of use. Instead, I chose to employ a long standing, and ongoing, scientific development as an exemplar. And so, I chose the ever evolving story of Thermodynamics as a scientific investigation at its best.
Conducted over a period of >200 years, Thermodynamics R&D, and application, benefitted from the highest levels of professionalism, collaboration, and technical thoroughness. New layers of application, methodology, and practice were made possible by the progressive advance of technology. In turn, this has seen measurement and modelling accuracy continually improved at a micro and macro level.
Perhaps most importantly, Thermodynamics rapidly became a primary tool in the advance of applied science/engineering/technology, spanning micro-tech, to aerospace and cosmology. I can think of no better a story to illustrate the breadth of scientific methodologies and applications at their best.
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.
ESR spectroscopy in liquid food and beverages.pptxPRIYANKA PATEL
With increasing population, people need to rely on packaged food stuffs. Packaging of food materials requires the preservation of food. There are various methods for the treatment of food to preserve them and irradiation treatment of food is one of them. It is the most common and the most harmless method for the food preservation as it does not alter the necessary micronutrients of food materials. Although irradiated food doesn’t cause any harm to the human health but still the quality assessment of food is required to provide consumers with necessary information about the food. ESR spectroscopy is the most sophisticated way to investigate the quality of the food and the free radicals induced during the processing of the food. ESR spin trapping technique is useful for the detection of highly unstable radicals in the food. The antioxidant capability of liquid food and beverages in mainly performed by spin trapping technique.
hematic appreciation test is a psychological assessment tool used to measure an individual's appreciation and understanding of specific themes or topics. This test helps to evaluate an individual's ability to connect different ideas and concepts within a given theme, as well as their overall comprehension and interpretation skills. The results of the test can provide valuable insights into an individual's cognitive abilities, creativity, and critical thinking skills
The ability to recreate computational results with minimal effort and actionable metrics provides a solid foundation for scientific research and software development. When people can replicate an analysis at the touch of a button using open-source software, open data, and methods to assess and compare proposals, it significantly eases verification of results, engagement with a diverse range of contributors, and progress. However, we have yet to fully achieve this; there are still many sociotechnical frictions.
Inspired by David Donoho's vision, this talk aims to revisit the three crucial pillars of frictionless reproducibility (data sharing, code sharing, and competitive challenges) with the perspective of deep software variability.
Our observation is that multiple layers — hardware, operating systems, third-party libraries, software versions, input data, compile-time options, and parameters — are subject to variability that exacerbates frictions but is also essential for achieving robust, generalizable results and fostering innovation. I will first review the literature, providing evidence of how the complex variability interactions across these layers affect qualitative and quantitative software properties, thereby complicating the reproduction and replication of scientific studies in various fields.
I will then present some software engineering and AI techniques that can support the strategic exploration of variability spaces. These include the use of abstractions and models (e.g., feature models), sampling strategies (e.g., uniform, random), cost-effective measurements (e.g., incremental build of software configurations), and dimensionality reduction methods (e.g., transfer learning, feature selection, software debloating).
I will finally argue that deep variability is both the problem and solution of frictionless reproducibility, calling the software science community to develop new methods and tools to manage variability and foster reproducibility in software systems.
Exposé invité Journées Nationales du GDR GPL 2024
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.
ESPP presentation to EU Waste Water Network, 4th June 2024 “EU policies driving nutrient removal and recycling
and the revised UWWTD (Urban Waste Water Treatment Directive)”
Describing and Interpreting an Immersive Learning Case with the Immersion Cub...Leonel Morgado
Current descriptions of immersive learning cases are often difficult or impossible to compare. This is due to a myriad of different options on what details to include, which aspects are relevant, and on the descriptive approaches employed. Also, these aspects often combine very specific details with more general guidelines or indicate intents and rationales without clarifying their implementation. In this paper we provide a method to describe immersive learning cases that is structured to enable comparisons, yet flexible enough to allow researchers and practitioners to decide which aspects to include. This method leverages a taxonomy that classifies educational aspects at three levels (uses, practices, and strategies) and then utilizes two frameworks, the Immersive Learning Brain and the Immersion Cube, to enable a structured description and interpretation of immersive learning cases. The method is then demonstrated on a published immersive learning case on training for wind turbine maintenance using virtual reality. Applying the method results in a structured artifact, the Immersive Learning Case Sheet, that tags the case with its proximal uses, practices, and strategies, and refines the free text case description to ensure that matching details are included. This contribution is thus a case description method in support of future comparative research of immersive learning cases. We then discuss how the resulting description and interpretation can be leveraged to change immersion learning cases, by enriching them (considering low-effort changes or additions) or innovating (exploring more challenging avenues of transformation). The method holds significant promise to support better-grounded research in immersive learning.
The debris of the ‘last major merger’ is dynamically youngSérgio Sacani
The Milky Way’s (MW) inner stellar halo contains an [Fe/H]-rich component with highly eccentric orbits, often referred to as the
‘last major merger.’ Hypotheses for the origin of this component include Gaia-Sausage/Enceladus (GSE), where the progenitor
collided with the MW proto-disc 8–11 Gyr ago, and the Virgo Radial Merger (VRM), where the progenitor collided with the
MW disc within the last 3 Gyr. These two scenarios make different predictions about observable structure in local phase space,
because the morphology of debris depends on how long it has had to phase mix. The recently identified phase-space folds in Gaia
DR3 have positive caustic velocities, making them fundamentally different than the phase-mixed chevrons found in simulations
at late times. Roughly 20 per cent of the stars in the prograde local stellar halo are associated with the observed caustics. Based
on a simple phase-mixing model, the observed number of caustics are consistent with a merger that occurred 1–2 Gyr ago.
We also compare the observed phase-space distribution to FIRE-2 Latte simulations of GSE-like mergers, using a quantitative
measurement of phase mixing (2D causticality). The observed local phase-space distribution best matches the simulated data
1–2 Gyr after collision, and certainly not later than 3 Gyr. This is further evidence that the progenitor of the ‘last major merger’
did not collide with the MW proto-disc at early times, as is thought for the GSE, but instead collided with the MW disc within
the last few Gyr, consistent with the body of work surrounding the VRM.
3. Why Count Cells?
• For maintaining cell cultures
Splitting cells or preparing for the next passage (usually
cells are diluted into a new culture flask with fresh media
for optimal growth)
• For preparing cells for transfection experiments
• For preparing cells for downstream experiments that require
accurate and consistent numbers of input cells,
including qPCR
5. Hemocytometer (Double Neubauer Ruled Metalized
Counting Chamber)
• Each counting chamber has a
mirrored surface with a 3 × 3 mm
grid of 9 counting squares
• The chambers have raised sides
that can hold a cover slip exactly 0.1
mm above the chamber floor
• Each of the 9 counting squares
holds a volume of 0.0001 mL (1 mm
x 1 mm x 0.1 mm)
• The average count in the squares
marked 1 to 4 in the figure,
multiplied by 10000 gives the cell
count/mL.
6. Cell Counting Using Hemocytometer:
• Remove bleached media from the T-flasks and rinse the cell
monolayer with Dulbecco’s Phosphate Buffered Saline (DPBS) without
calcium or magnesium.
• Remove DPBS and add trypsin-EDTA solution (1 mL for T-25 flasks and
3 mL for T-75 flasks) to the flasks followed by incubation at 37°C, to
dissociate the cells from the adhering surface
• When the cells appeared to be detached, add complete growth media
to neutralize the trypsin in volumes that were double that of the
trypsin-EDTA used for dissociation
• >95% of the cells should be single cells
• After mixing the cell suspension to ensure uniform distribution of
cells, load 10 μL of the cell suspension into the counting chamber.
• Place Neubauer chamber was placed under an inverted microscope
and view the cells at 100 x magnification
• Count the cells in quadrants labeled 1, 2, 3 and 4 and multiply the
average value by 10000 to obtain the number of cells per mL.
8. Automated Cell Counting – Flow Cytometry
• If suitable dyes are used, viability also can be assessed
• Acridine Orange (AO)- Cell membrane permeable Stains nucleus
• Propidium Iodide (PI)- Impermeable
9. Automated Cell Counting – Spectrophotometry
• Not very reliable
• More cells More turbidity High OD
• Relative count
• Absolute count: When you have a sample with known cell number
• Not suitable if media is turbid
11. Cell Viability Assays:
• For maintaining cell cultures
Splitting cells or preparing for the next passage (usually cells are
diluted into a new culture flask with fresh media for optimal
growth)
• For preparing cells for transfection experiments
• For preparing cells for downstream experiments that require
accurate and consistent numbers of input cells, including qPCR
• To assess toxic effect of drugs/ chemicals
• After cryopreservation
12. Cell Viability Assays
• Non-fluorescence based
Trypan Blue
Erythrosin B
MTT
XTT
• Fluorescence based
• Chemiluminescence based
13. Trypan Blue Exclusion Test
• Live cells possess intact cell membranes that exclude certain dyes
such as trypan blue, whereas dead cells do not
• Add 10 μL of 0.4% trypan blue to 10 μL of the cell suspension
• After proper mixing of the dye and the cell suspension, load 10 μL
of the mixture into the counting chamber
• Viable cells are characterized by a clear cytoplasm whereas
nonviable cells possess a blue cytoplasm
• Count the viable cells in quadrants 1 to 4 of the Neubauer chamber
within 5 minutes and multiply the average of this value by 10000 x
dilution factor (2, in this case)
• This gives the number of viable cells per mL of the cell suspension
• This can also be expressed as a percentage of the total number of
cells.
14. Trypan Blue vs Erythrosin B
• Trypan Blue
Incubation: 2-5 minutes
Binds to serum proteins
Less clear background
Potential carcinogen
• Erythrosin B
No incubation
No binding
Clear background
Less toxic
15. MTT Assay
• 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
• Reduction of yellow MTT by mitochondrial succinate
dehydrogenase yields insoluble dark purple formazan
• The cells are then solubilised with an organic solvent (e.g. DMSO)
and the released, solubilised formazan reagent is measured
spectrophotometrically at 570 nm using a micro-titer plate reader
16. MTT Assay:
• Prepare the cell suspension and count the cells
• Plate the cells onto a 96-well tissue culture plate, with seeding
densities of 1x106, 1x105, 1x104, and 1x103 cells/mL
• Make dilutions so as to maintain 200μL of complete media/well
and seed the wells in hexaplicate
• Only the inner rows and columns of the plate are to be used so as
to minimize cell growth variations due to different medium
evaporation rates at the periphery
• One well should be maintained as blank, in which only media is
added
• Incubate the tissue culture plate in a CO2 incubator at 37°C
overnight.
• Check the cell growth on the next day
17. MTT Assay:
• Remove the bleached media from each well and add 100 μL of MTT
(1 mg/mL) diluted in DPBS to each well
• Incubate at 37°C for 4 hours, remove the supernatant and add
100μL of DMSO to each well
• Incubate the plate in the dark for 60 minutes and measure the
absorbance using a micro-titer plate reader at 570 nm
• Calculate the mean absorbance and plot it against the number of
cells/mL.
19. Luminescence Based Detection
• Detects presence of ATP
• When cells lose membrane integrity, they lose the ability to
synthesize ATP and endogenous ATPases rapidly deplete any
remaining ATP from the cytoplasm
• Highly sensitive
20. Fluorescence based Detection:
• Live cells contain esterases
• Non-fluorescent substrates Fluorescent molecules
• Intact intracellular membrane retains the cleaved
fluorescent products inside the cell
• Dead cells, are deficient in esterase activity and their
compromised membranes lead to substrate leaks from
cells