Fluorescence activated cell sorting (FACS) enables the separation of different cell populations from a mixed community by sorting individual cells based on their light scattering and fluorescent characteristics. Cells are tagged with fluorescent dyes and passed through a stream that is broken into droplets and charged before cells are deflected into collection tubes. FACS has applications in separating cell types like γδ T cells, screening proteins, and determining biomarker levels. While powerful, FACS also has limitations such as slow speed, high cost, and a need for specialized equipment.
Fluorescence- Activated Cell Sorter is a powerful technique used in cell sorting, cell-cycle analysis etc.
The presentation gives a basic understanding of the principle of FACS, instrumentation, interpretation of results, applications, how to do cell-cycle analysis using FACS and various troubleshooting tips.
Fluorescence- Activated Cell Sorter is a powerful technique used in cell sorting, cell-cycle analysis etc.
The presentation gives a basic understanding of the principle of FACS, instrumentation, interpretation of results, applications, how to do cell-cycle analysis using FACS and various troubleshooting tips.
CLONAL SELECTION THEORY IS AN SCIENTIFIC THEORY IN IMMUNOLOGY THAT EXPALINS THE FUNCTION OF CELLS OF THE IMMUNE SYSTEM IN RESPONSE TO SPECIFIC ANTIGEN INVADING THE BODY.
This is technique used widely for protein separation from a mixture and is very easy and less costly method. Slides cover all essential points about EMSA and it is quite interesting to know that how it detect and separate different proteins and their mobility shift assay.
Gel electrophoresis native, denaturing&reducingLovnish Thakur
Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation.
Compare density gradient centrifugation, Magnet-activated cell sorting (MACS), and Fluorescence-activated cell sorting (FACS) in the isolation of pure stem cell populations from a heterogeneous suspension. Discuss the advantages and disadvantages of each technology as well as the emerging (recent) methods in stem cells separation.
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CLONAL SELECTION THEORY IS AN SCIENTIFIC THEORY IN IMMUNOLOGY THAT EXPALINS THE FUNCTION OF CELLS OF THE IMMUNE SYSTEM IN RESPONSE TO SPECIFIC ANTIGEN INVADING THE BODY.
This is technique used widely for protein separation from a mixture and is very easy and less costly method. Slides cover all essential points about EMSA and it is quite interesting to know that how it detect and separate different proteins and their mobility shift assay.
Gel electrophoresis native, denaturing&reducingLovnish Thakur
Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation.
Compare density gradient centrifugation, Magnet-activated cell sorting (MACS), and Fluorescence-activated cell sorting (FACS) in the isolation of pure stem cell populations from a heterogeneous suspension. Discuss the advantages and disadvantages of each technology as well as the emerging (recent) methods in stem cells separation.
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CHI's Targeting Stromal Cells in Cancer and Inflammatory Diseases Conference ...James Prudhomme
This virtual meeting will highlight cutting-edge science and provide insight into recent developments towards therapeutic stromal cell targeting in cancer and chronic inflammatory diseases. View full details and register: https://www.healthtech.com/stroma-conference
Compare density gradient centrifugation, magnet-activated cell sorting
(MACS), and fluorescence-activated cell sorting (FACS) in the isolation of pure stem cell populations from a heterogeneous suspension
mge_a4_study_q4.pdf
ORIGINAL RESEARCH
published: 12 October 2015
doi: 10.3389/fmicb.2015.01063
Edited by:
Miklos Fuzi,
Semmelweis University, Hungary
Reviewed by:
Atte Von Wright,
University of Eastern Finland, Finland
Dmitri Debabov,
NovaBay Pharmaceuticals, USA
*Correspondence:
Anne-Brit Kolstø,
Laboratory for Microbial Dynamics,
Department of Pharmaceutical
Biosciences, School of Pharmacy,
University of Oslo, Postbox 1068
Blindern, 0316 Oslo, Norway
[email protected]
†Present address:
Roger Simm,
Norwegian Veterinary Institute, Oslo,
Norway;
Massoud Saidijam,
Research Centre for Molecular
Medicine, Department of Molecular
Medicine and Genetics, School
of Medicine, Hamadan University
of Medical Sciences, Hamadan, Iran
Specialty section:
This article was submitted to
Antimicrobials, Resistance
and Chemotherapy,
a section of the journal
Frontiers in Microbiology
Received: 17 August 2015
Accepted: 15 September 2015
Published: 12 October 2015
Citation:
Kroeger JK, Hassan K, Vörös A,
Simm R, Saidijam M, Bettaney KE,
Bechthold A, Paulsen IT,
Henderson PJF and Kolstø A-B
(2015) Bacillus cereus efflux protein
BC3310 – a multidrug transporter
of the unknown major facilitator family,
UMF-2. Front. Microbiol. 6:1063.
doi: 10.3389/fmicb.2015.01063
Bacillus cereus efflux protein
BC3310 – a multidrug transporter of
the unknown major facilitator family,
UMF-2
Jasmin K. Kroeger1,2, Karl Hassan3, Aniko Vörös1, Roger Simm1†, Massoud Saidijam4†,
Kim E. Bettaney4, Andreas Bechthold2, Ian T. Paulsen3, Peter J. F. Henderson4 and
Anne-Brit Kolstø1*
1 Laboratory for Microbial Dynamics, Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo,
Oslo, Norway, 2 Institut für Pharmazeutische Biologie und Biotechnologie, Albert-Ludwigs Universität, Freiburg, Germany,
3 Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia, 4 School of
BioMedical Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK
Phylogenetic classification divides the major facilitator superfamily (MFS) into 82 families,
including 25 families that are comprised of transporters with no characterized functions.
This study describes functional data for BC3310 from Bacillus cereus ATCC 14579, a
member of the “unknown major facilitator family-2” (UMF-2). BC3310 was shown to
be a multidrug efflux pump conferring resistance to ethidium bromide, SDS and silver
nitrate when heterologously expressed in Escherichia coli DH5α �acrAB. A conserved
aspartate residue (D105) in putative transmembrane helix 4 was identified, which was
essential for the energy dependent ethidium bromide efflux by BC3310. Transport
proteins of the MFS comprise specific sequence motifs. Sequence analysis of UMF-
2 proteins revealed that they carry a variant of the MFS motif A, which may be used
as a marker to distinguish easily between this family and other MFS proteins. Genes
orthologous to bc3310 are hig.
Review of literature on carbon nanotubes exposure risks to human health, and hypes involved in exageration of risks and raising issues regarding research carried out in unrealistic laboratory environment that exhibit excess of human uptake.
Medcrave Group - Microfluidic technologiesMedCrave
Exosomes are cell-released small membrane vesicles derived from the endolysosomal pathway with a size range of 30-150 nm. Since the first discovery in 1981, exosomes have been found to be released from various cell types and present in many biological fluids, including blood, urine, erebrospinal fluid and ascites. Significant attention has been focused on exosome molecular components (e.g. roteins, mRNA and miRNA) which have been implicated in a variety of physiological functions and pathological disease states.
A statistical framework for multiparameter analysis at the single cell levelShashaanka Ashili
Phenotypic characterization of individual cells provides crucial insights into intercellular heterogeneity and enables access to information that is unavailable from ensemble averaged, bulk cell analyses. Single-cell studies have attracted significant interest in recent years and spurred the development of a variety of commercially available and research-grade technologies. To quantify cell-to-cell variability of cell populations, we have developed an experimental platform for real-time measurements of oxygen consumption (OC) kinetics at the single-cell level. Unique challenges inherent to these single-cell measurements arise, and no existing data analysis
methodology is available to address them. Here we present a data processing and analysis method that addresses challenges encountered with this unique type of data in order to extract biologically relevant information. We applied the method to analyze OC profiles obtained with single cells of two different cell lines derived from metaplastic and dysplastic human Barrett’s esophageal epithelium. In terms of method development, three main challenges were considered for this heterogeneous dynamic system: (i) high levels of noise, (ii) the lack of a priori knowledge of single-cell dynamics, and (iii) the role of intercellular variability within and across cell types.
Several strategies and solutions to address each of these three challenges are presented. The features such as slopes, intercepts, breakpoint or change-point were extracted for every OC profile and compared across individual cells and cell types. The results demonstrated that the extracted features facilitated exposition of subtle differences between individual cells and their responses to
cell–cell interactions. With minor modifications, this method can be used to process and analyze
data from other acquisition and experimental modalities at the single-cell level, providing a valuable statistical framework for single-cell analysis.
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
Exosomes biomarkers mediating important biological process,especially in the systemic disease
diagnostics and therapeutics,yet the protective exosomal vesicle structure hinders rapid,simple detection of the harbored molecules.
UiPath Test Automation using UiPath Test Suite series, part 3DianaGray10
Welcome to UiPath Test Automation using UiPath Test Suite series part 3. In this session, we will cover desktop automation along with UI automation.
Topics covered:
UI automation Introduction,
UI automation Sample
Desktop automation flow
Pradeep Chinnala, Senior Consultant Automation Developer @WonderBotz and UiPath MVP
Deepak Rai, Automation Practice Lead, Boundaryless Group and UiPath MVP
The Art of the Pitch: WordPress Relationships and SalesLaura Byrne
Clients don’t know what they don’t know. What web solutions are right for them? How does WordPress come into the picture? How do you make sure you understand scope and timeline? What do you do if sometime changes?
All these questions and more will be explored as we talk about matching clients’ needs with what your agency offers without pulling teeth or pulling your hair out. Practical tips, and strategies for successful relationship building that leads to closing the deal.
Kubernetes & AI - Beauty and the Beast !?! @KCD Istanbul 2024Tobias Schneck
As AI technology is pushing into IT I was wondering myself, as an “infrastructure container kubernetes guy”, how get this fancy AI technology get managed from an infrastructure operational view? Is it possible to apply our lovely cloud native principals as well? What benefit’s both technologies could bring to each other?
Let me take this questions and provide you a short journey through existing deployment models and use cases for AI software. On practical examples, we discuss what cloud/on-premise strategy we may need for applying it to our own infrastructure to get it to work from an enterprise perspective. I want to give an overview about infrastructure requirements and technologies, what could be beneficial or limiting your AI use cases in an enterprise environment. An interactive Demo will give you some insides, what approaches I got already working for real.
Encryption in Microsoft 365 - ExpertsLive Netherlands 2024Albert Hoitingh
In this session I delve into the encryption technology used in Microsoft 365 and Microsoft Purview. Including the concepts of Customer Key and Double Key Encryption.
Connector Corner: Automate dynamic content and events by pushing a buttonDianaGray10
Here is something new! In our next Connector Corner webinar, we will demonstrate how you can use a single workflow to:
Create a campaign using Mailchimp with merge tags/fields
Send an interactive Slack channel message (using buttons)
Have the message received by managers and peers along with a test email for review
But there’s more:
In a second workflow supporting the same use case, you’ll see:
Your campaign sent to target colleagues for approval
If the “Approve” button is clicked, a Jira/Zendesk ticket is created for the marketing design team
But—if the “Reject” button is pushed, colleagues will be alerted via Slack message
Join us to learn more about this new, human-in-the-loop capability, brought to you by Integration Service connectors.
And...
Speakers:
Akshay Agnihotri, Product Manager
Charlie Greenberg, Host
GraphRAG is All You need? LLM & Knowledge GraphGuy Korland
Guy Korland, CEO and Co-founder of FalkorDB, will review two articles on the integration of language models with knowledge graphs.
1. Unifying Large Language Models and Knowledge Graphs: A Roadmap.
https://arxiv.org/abs/2306.08302
2. Microsoft Research's GraphRAG paper and a review paper on various uses of knowledge graphs:
https://www.microsoft.com/en-us/research/blog/graphrag-unlocking-llm-discovery-on-narrative-private-data/
Builder.ai Founder Sachin Dev Duggal's Strategic Approach to Create an Innova...Ramesh Iyer
In today's fast-changing business world, Companies that adapt and embrace new ideas often need help to keep up with the competition. However, fostering a culture of innovation takes much work. It takes vision, leadership and willingness to take risks in the right proportion. Sachin Dev Duggal, co-founder of Builder.ai, has perfected the art of this balance, creating a company culture where creativity and growth are nurtured at each stage.
Key Trends Shaping the Future of Infrastructure.pdfCheryl Hung
Keynote at DIGIT West Expo, Glasgow on 29 May 2024.
Cheryl Hung, ochery.com
Sr Director, Infrastructure Ecosystem, Arm.
The key trends across hardware, cloud and open-source; exploring how these areas are likely to mature and develop over the short and long-term, and then considering how organisations can position themselves to adapt and thrive.
Key Trends Shaping the Future of Infrastructure.pdf
Application of Fluorescence Activated-Cell Sorting (FACS) in separation of different populations of cells from a mixed community
1. Application of Fluorescence Activated-Cell
Sorting (FACS) -Separation of different
populations of cells from a mixed
community
Presented by :
Goh Mei Ying (0317999)
Lim Tze Shien (0323020)
Muhammad Uzair (0321618)
Nur Nabihah Mohamat (0318664)
Ting Sing Hong (0317799)
2. Introduction
Fluorescence Activated-Cell Sorting
Enables a mixture of different cells to be sorted one by one into one or more
containers.
Cells are sorted according to their specific light scattering and fluorescent
characteristics (Robertson 2010).
(Blogs.sun.ac.za n.d.)
The ways to display the FACS data collected by computer:
(Robertson 2010)
3. (Bio.davidson.edu 2001)
Cells (by tagging to an antibody linked to fluorescent dye) are passed into
the middle of a fast flowing liquid stream
Vibration is applied to break the stream up into droplets
Laser light excites the dye which emits a colour of light
The colored light is detected by a photomultiplier tube or a light detector
An electrical charging ring is positioned
An electrical charge is applied to the stream and the newly formed drop will
form with charge
Charged drop will be then deflected left or right by charged electrodes and
into waiting sample tube (Robertson 2010)
4. Discussion
Application
used to separate different populations of cells from a mixed community where it
allows physical enrichment or isolation even of yet uncultured organisms that
can be used for subsequent molecular genetic studies and cultivation (Roest
2007).
Park et al. (cited in Roest 2007) used this technique for analysis of activated-
sludge and Yellowstone Lake hydrothermal vent samples and he detected
various unknown bacterial species which were not detectable in the previous
original sample due to low relative abundance and this limitation could be
overcome by the application of FACS.
5. Discussion
Application
Separation & isolation of γδ T cells from a population of lymphocytes.
Why study γδ T cells ?
Deficiency of γδ T cells aggravates colitis in animal models.
Suggesting that they possesses regulatory properties in secretion of cytokines.
γδ T cells specific subset of T cells, play a prominent role in recognizing
lipid antigens, may be triggered by alarm signals, such as heat shock proteins.
Cell sample are stained with fluorescent antibody which binds specifically to
surface markers on γδ T cells.
(Kühl et al. 2009)
(Dartmouth Undergraduate Journal of Science 2008)
6. Discussion
Application
Screening for intracellular and secreted proteins
Cells with high secretion
rate can be sorted and
subcloned.
Plasmids are isolated and
retransformed into the host
strain.
(Mattanovich & Borth 2006)
7. Idiopathic Pulmonary Fibrosis (IPF)
Diagnostics
Idiopathic – any disease/condition that arise spontaneously with an
unknown cause.
Pulmonary Fibrosis – scar tissue forms in lungs.
Lung tissue become stiff and scarred.
S & S – dry cough & trouble breathing.
8. Idiopathic Pulmonary Fibrosis (IPF)
Diagnostics
Myeloid-Derived Suppressor Cells (MDSC)
Suppress immune system
Tissue remodeling angiogenesis aggressive cancer
(Bronte and Gabrilovich 2016)
Fluorescence Activated Cell Sorting (FACS) helps to determine quantity of
MDSC in blood samples.
Higher number of MDSC found in IPF patients.
MDSC as biomarkers.
(Lawrence 2016)
9. Sorter is mainly for the use on a large population of cells causing the recovery rate is fairly slow (Lauren 2007).
There is a tradeoff between the sorting speed, purity rate and recovery state (Lauren 2007).
Currently, traditional FACS machine is very large and space consuming but it is the ideal size for its function (Lauren
2007).
In modern cancer and immunology research, it is considered expensive due to the inevitable process to recover all the
cells from the sorter with maximum possible purity rate (FACS 2016).
FACS is limited to its specific purpose and prolonged process, more than 6 hours compared to magnetic cell sorting
type (eg. FACS only enable one cell to pass through the laser focus at a time) (Catherine, Brian T and Timothy C 2010).
Requires debulking process first (Catherine, Brian T and Timothy C 2010).
Limited number of fluorophore-conjugated antibody reagent for clinical processing (Catherine, Brian T and Timothy
C 2010).
Direct and indirect method for Immunofluorescence are correlated to each other to give a better results (Abcam.com
2016).
Challenging to detect low abundance proteins even with indirect methods. (eg. Biotinylated antibodies offer an extra
layer for increased signal amplification.) (Abcam.com 2016).
Main Challenges & Limitation
10. Table 1: Comparison of cell isolation approaches by surface antigen-based
Figure 1:Traditional cell sorting machine
(Lauren 2007).
11. Conclusion
Function of FACS To sort cells according to their specific light scattering and fluorescent characteristics.
Applications
Separation of different populations of cells from a mixed community
Separation & isolation of γδ T cells from a population of lymphocytes
Screening of intracellular and secreted protein
Current development
Determination of quantity of MDSC in blood samples of IPF patients.
Challenges & limitation
Slow recovery rate
Space consuming
Expensive; Long processing hours
Requires debulking process
Limited number of fluorophore-conjugated antibody reagent
Challenging to detect low abundance proteins
12. References
Abcam.com 2016, Direct vs indirect immunofluorescence | Abcam., online, viewed 24 September 2016, <http://www.abcam.com/secondary-
antibodies/direct-vs-indirect-immunofluorescence>.
Bio.davidson.edu 2001, FACS Methodology, viewed 21 September 2016, <http://www.bio.davidson.edu/courses/genomics/method/facs.html>.
Blogs.sun.ac.za n.d., BD FACS Calibur », viewed 21 September 2016, <http://blogs.sun.ac.za/fmc/bd-facs-calibur/>.
Bronte, V. and Gabrilovich, D. 2016, Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards.
Nature Communications, 7, p.12150.
Catherine, M., Brian T, F. and Timothy C, F. 2010, Fluorescence-Activated Cell Sorting for CGMP Processing of Therapeutic Cells. 1st ed.
[ebook] Sparks: BD biosciences, p.7, viewed 23 September 2016,
<https://www.researchgate.net/profile/Timothy_Fong2/publication/228470167_FluorescenceActivated_Cell_Sorting_for_CGMP_Processing_of
_Therapeutic_Cells/links/55f6ef6e08ae07629dbb159e.pdf>.
Dartmouth Undergraduate Journal of Science 2008, 488 nm: A Review of FACS Technology and its Application in Biological Research, online,
viewed 23 September 2016, < http://dujs.dartmouth.edu/2008/02/488-nm-a-review-of-facs-technology-and-its-application-in-biological-
research/#.V-s5GFt96Un>.
FACS, M. 2016, Magnetic-activated cell sorting vs. FACS. [online] Biology.stackexchange.com, viewed 22 September 2016,
<http://biology.stackexchange.com/questions/31482/magnetic-activated-cell-sorting-vs-facs>.
Kühl, A. A., Pawlowski, N. N., Grollich, K., Blessenohl, M., Westermann, J., Zeitz, M., Loddenkemper, C. & Hoffmann, J. C. 2009, ‘Human
peripheral γδ T cells possess regulatory potential’, Immunology, 128(4), 580–588. http://doi.org/10.1111/j.1365-2567.2009.03162.x
13. Lauren, N. 2007, Fluorescence-Activated Cell Sorting in Microfluidic Devices. 1st ed. [ebook] Boulder: University of Colorado, p.13, viewed 24
September 2016, <http://www.colorado.edu/physics/Web/reu/Projects/Projects%202007/Lauren%20Nicolaisen.pdf>.
Lawrence, J. 2016, Scientists Discover Biomarkers for Diagnosing Idiopathic Pulmonary Fibrosis, online, Naturalsciencenews.com, viewed 28
September 2016, <http://naturalsciencenews.com/2016/09/02/scientists-discover-biomarkers-for-diagnosing-idiopathic-pulmonary-fibrosis/>.
Mattanovich, D. & Borth, N. 2006, ‘Applications of cell sorting in biotechnology’, Microbial Cell Factories, 5, 12. http://doi.org/10.1186/1475-
2859-5-12
Robertson, S. 2010, Fluorescence-Activated Cell Sorting, viewed 21 September 2016, < http://www.news-medical.net/life-
sciences/Fluorescence-Activated-Cell-Sorting.aspx>.
Roest, K. 2007, ‘Microbial community analysis in sludge of anaerobic wastewater treatment systems’, PhD thesis, Wageningen University,
Netherlands.
References
Fluorescence-Activated Cell Sorting is a flow cytometer that enables a mixture of different cells to be sorted one by one into one or more containers according to their specific light scattering and fluorescent characteristics. Here are the ways to display the data. First method is best to display the data if no cells are labeled both colors. For the second methods, we cant to count for each spot, just can see the relative density. Top right: no cells labelled both colors, bottom left: cells that unlabelled.
Cells(by tagging to an antibody linked to fluorescent dye) are passed into the middle of a fast flowing liquid stream to ensure that they are well separated.
Vibration is applied to break the stream up into droplets in such a way one cell is presented per drop.
Laser light excites the dye which emits a color of light
The colored light is detected by a photomultiplier tube or a light detector
An electrical charging ring is positioned
An electrical charge is applied to the stream and the newly formed drop will form with charge
Charged drop will be then deflected left or right by charged electrodes and into waiting sample tube
Deficiency in γδ T cells aggravates colitis in animal models suggesting that γδ T cells have regulatory properties. Therefore, proliferation, suppression and cytokine secretion of human γδ T cells were determined in vitro.
When sorting is used for the selection of over-producing cells, there are three main objectives:
to reduce the work load necessary to find over-producers,
(2) to reduce the time required and
(3) to find the cells with the highest possible production rates, ideally combined with other advantageous properties
Immunofluorescence based screening methods for intracellular and secreted proteins.
A: Screening for secreted proteins, developed for mammalian cells. A product specific catching antibody is immobilized on the cell surface, which binds an amount of product proportional to the secretion rate. After staining with a product specific antibody and a secondary antibody, cells with high secretion rate can be sorted and subcloned.
B: Screening system for intracellular, plasmid encoded proteins, developed for E. coli. Cells are fixed with ethanol and stained with a product specific antibody and a secondary antibody After sorting, plasmids are isolated and retransformed into the host strain.
