Flow Cytometry

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8 Stimulating frozen PBMCs?
By Christine Mall · University of California, Davis

Lenin Godoy · Ponce School of Medicine and Health Sciences

Completely agree with the freezing media. We use RPMI-FBS 50%, 10% DMSO. Primary cells are very delicate so we also use a thawing medium for thaw th... [more]

Completely agree with the freezing media. We use RPMI-FBS 50%, 10% DMSO. Primary cells are very delicate so we also use a thawing medium for thaw the cells. 10% Dextrose, 40% RPMI-1640 and 50%FBS.

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4 Can I use a different cell lines as negative or positive controls when doing flow cytometry?
By Fei Yang · Huazhong University of Science and Technology

Xiaoyan Li

I agree with Elvis Kidzeru . firstly, different cells have different forward and side light-scatter characteristics,which will affect on the instrume... [more]

I agree with Elvis Kidzeru . firstly, different cells have different forward and side light-scatter characteristics,which will affect on the instrument setting; Secondly, both IFNg and Granzyme B perform intracellular staining ,usually positive cells are very small population in this case, so performer must set strict controls,using the same cells as control for single color staining and fluorescence minus one (FMO) controlsbased on the same cells for multicolor staining .

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Question:
New Is there any way to measure mevalonate pathway metabolites by flow cytometry in human monocytes
We wonder whether inflammasome activation results in modulation of the mevalonate pathway (especially IPP levels). Reports have described the effect o... [more]

We wonder whether inflammasome activation results in modulation of the mevalonate pathway (especially IPP levels). Reports have described the effect of mevalonate pathway on triggering inflammasome activation but not vice versa. In particular we are interested to look for modulations in this pathway in FMF patients who carry mutations in MEFV gene encoding for pyrin.

By Alberta Paul · Ondokuz Mayıs Üniversitesi

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36 Inexpensive method for assessment of the stages of apoptosis using flow cytometry?
By Michael Fenn · University of Florida

Hongjuan Cui · Southwest University in Chongqing

I always use Annexin V and &-AAD for flow cytometry, and I always get what I expected. Good luck.

I always use Annexin V and &-AAD for flow cytometry, and I always get what I expected. Good luck.

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3 Should I expect poor CD4 staining of NHP PBMCs following expansion with anti-CD3/anti-CD28 and subsequent stimulation with PMA/Ionomycin?
By Matthew Auten · Louisiana State University Health Sciences Center New Orleans

Anthony Park · Monash University

As the other two answers have said, yes CD4 will be internalised following stimulation (as will CD8 and I think CD3 also). However, if you fix and per... [more]

As the other two answers have said, yes CD4 will be internalised following stimulation (as will CD8 and I think CD3 also). However, if you fix and perm your cells and then stain you will be able to identify the CD4+ cells.

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6 Is it possible to perform cell cycle analysis on FACSAria?
By kamariah ibrahim · National University of Malaysia

kamariah ibrahim · National University of Malaysia

Thanks for your suggestions. Will try to troubleshoot and inform to you later. =)

Thanks for your suggestions. Will try to troubleshoot and inform to you later. =)

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30 Whether PI+ / Annexin V-FITC+ cells are classified as LateApoptotic cells or Necrotic cells?
By Amir Tayaranian · University of Tehran

Michela Perego · Wistar Institute

for late apoptosis you should use caspase 3 and/or TUNEL assay, otherwise you should get trouble publishing your data. be careful with compensation wi... [more]

for late apoptosis you should use caspase 3 and/or TUNEL assay, otherwise you should get trouble publishing your data. be careful with compensation with PI too...

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6 Flow cytometry question
By Mia Shandell · Columbia University

Leonardo Chicaybam · Brazilian National Cancer Institute

Maybe it is the transfection procedure. After the transfection the morphology of cells may change a little bit, leading to a different backgroung fluo... [more]

Maybe it is the transfection procedure. After the transfection the morphology of cells may change a little bit, leading to a different backgroung fluorescence. I have seen this with electroporation of T lymphocytes and lentivirus transduction of 293T cells.

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1 Can a flow cytometer be used to quantify specific proteins? Has it been done in sperm?
By Zarka Daniel · Spanish National Research Council

Hani Harb · Philipps-Universität Marburg

Well, as far as i know, you can stain different proteins intracellularly if the right staining there. In my group, we measure cytokine levels through ... [more]

Well, as far as i know, you can stain different proteins intracellularly if the right staining there. In my group, we measure cytokine levels through flow cytometry but in the supernatants of different cells after stimulation. An intracellular staining maybe difficult to be done for many proteins at once.

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3 Use of CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit for detecting apoptosis: Difficultly in getting expected results
By Michael Fenn · University of Florida

Michael Fenn · University of Florida

Daniel Thank you very much, that is extremely helpful, I am fairly new to flow cytometry data analysis and using FCS Express 4 software, so I am not e... [more]

Daniel Thank you very much, that is extremely helpful, I am fairly new to flow cytometry data analysis and using FCS Express 4 software, so I am not exactly sure about how to properly move the axis, or how to adjust compensation. But I agree, from what you say it seems to make sense. Very timely too, as I was just going through this flow data. I actually did some fluorescence microscopy of live cells stained with this assay today, and could see that obviously the cell treated with the different anti-cancer agents were fluorescing, but he image quality was a rather poor. The problem seemed to be that the ex/em filter cubes on the microscope set up I was using does not seem to be optimal for Caspase dye (using DAPI/GFP/TRITC fluorescence block set up). So, I removed the imaging buffer and quickly added some 3.7% PFA to fix them so I might hopefully look at them on another scope in the next few days with a more optimal set up. But either way, the Caspase 3/7 stain is working to some extent, I just need to modify my protocol and my flow analysis. Thank you again for your very helpful insight!!

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6 Lysotracker to follow autophagy
By Patrice Petit · Université René Descartes - Paris 5

The Duy Nguyen · Goethe-Universität Frankfurt am Main

Dear Patrice, in our lab we are using Lyso-ID® Red detection kit (GFP-Certified®) from Enzo Life Sciences and it works very well with flow cytometr... [more]

Dear Patrice, in our lab we are using Lyso-ID® Red detection kit (GFP-Certified®) from Enzo Life Sciences and it works very well with flow cytometry. Best, The Duy

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12 FACS Buffer: Why include FBS?
By Kenneth Gareau · Excelimmune, Inc.

Martin Waterfall · The University of Edinburgh

As already stated the addition of protein (FBS or BSA) helps reduce non-specific antibody interactions and cell aggregation. I believe they also help ... [more]

As already stated the addition of protein (FBS or BSA) helps reduce non-specific antibody interactions and cell aggregation. I believe they also help with maintaining osmolarity of the buffer and overall cell viability. Azide is a metabolic inhibitor and is added for several reasons : 1. prevent bacterial/fungal growth 2. allows storage of buffer and staining at room temperature 3. inhibits capping, antigen internalisation and shedding of surface antigens 4. inhibits antibody internalisation Avoid azide supplemented media if subsequent cell function is required, in which case filter PBS supplemented with 0.1-2% FCS(or BSA - generally has significantly lower associated autofluorescence ) and use cold for staining steps to mitigate 3 and 4

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9 Does anyone have a Flow Cytometry protocol for spheroids?
By Elaine Silveira · University of São Paulo

Meghan Cuddihy · University of Michigan

I know this team is working with flow cytometry of whole spheroids (link is to their poster): http://3dbiomatrix.com/wp-content/uploads/2012/07/cyto20... [more]

I know this team is working with flow cytometry of whole spheroids (link is to their poster): http://3dbiomatrix.com/wp-content/uploads/2012/07/cyto2012-poster.jpg I'm not sure of the final details of the work, but you can always contact them or the company making the flow cytometer. I hope this is helpful!

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5 Sorting Treg cells from HIV patients.
By Cristina Peligero · University Pompeu Fabra

Rachel Hewitt · Medical Research Council (UK)

Hi Cristina, if your experiment were a flow cytometry based proliferation assay then you would not need to start with a 'pure' Treg population, unless... [more]

Hi Cristina, if your experiment were a flow cytometry based proliferation assay then you would not need to start with a 'pure' Treg population, unless you were worried about other cell interactions, it could save quite a bit of hassle. You could separate your Treg target population based on combinations of the markers suggested in several of the other answers. If for example you were to perform a CFSE proliferation assay then you would also be able to stain for phenotypic surface markers and also permeabilize the cells for intracellular markers such as FoxP3 etc at the end of the assay before running the samples, gate Tregs in and non Tregs out etc. in the analysis. As for the combinations of markers, it might be a case of trial and error to see what actually works best for your samples. Hope this helped, Rachel.

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7 Will staining splenocytes with MHC I and II tetramers at the same time affect the binding of either of the tetramers?
By Shailendra Tallapaka · University of Nebraska Medical Center

Jeffrey Frelinger · The University of Arizona

You should note that the best protocls for class I and class II tetramer binding differ-generally class I tetramers bind better, ut class II seem to n... [more]

You should note that the best protocls for class I and class II tetramer binding differ-generally class I tetramers bind better, ut class II seem to need more time- maybe to endocytose the TCR:tetramer complex to be stable staining, so usually done at 37 for 60 min while class I binding is faster and usually done at RT or 4. No matter what the contyrols noted aboe by Matthew should be done for every tetramer pair. Also remember that the freqeuncy of CD4/Tet+ cells is likely to be a lotl ower than CD8/tet+ cells.

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3 Do fluorophore-conjugated antibodies attatch to plastic?
By Maria Garcia-Leon · Universidad Autónoma de Madrid

Dmitry Kazansky · N.N. Blokhin Cancer Research Center

Good luck in your studies, Maria!

Good luck in your studies, Maria!

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1 Renal macrophage isolation.
By Ahmed Kamal · University of Alabama at Birmingham

Juan Spera · National University of General San Martín

I´ve had serious problems with the Microbeads CD11b from Miltenyi. I noticed that purified splenic CD11b cells are useless for functional assays, so ... [more]

I´ve had serious problems with the Microbeads CD11b from Miltenyi. I noticed that purified splenic CD11b cells are useless for functional assays, so it seems that the purification procedure irreversiblely alters these cells. Maybe you should try the negative selection kit for purifying CD11b from a Canadian company (www.stemcell.com). Negative selection is always better cos you don´t trigger any signal on your target cell. If your Miltenyi Microbeads are close to expiration date you should try to use twice or three times more antibodies than the protocol says (30ul instead of 10ul for 10million cells).

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3 Sorting and counting of dendritic cells.
By Guruprasad Gopalakrishnan · PSG Institute of Medical Sciences & Research

Dario Leone · Medical University of Vienna

From peripheral human blood you can easy sort out also a third populatiion that express CD141 and is suppost to be the CD8 mouse equivalent

From peripheral human blood you can easy sort out also a third populatiion that express CD141 and is suppost to be the CD8 mouse equivalent

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6 Use of CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit for detecting apoptosis: Difficultly in getting expected results
By Michael Fenn · University of Florida

Michael Fenn · University of Florida

Yes, those are options which are also already being explored, but the idea is to be able to compare as many of the most common methods as possible, so... [more]

Yes, those are options which are also already being explored, but the idea is to be able to compare as many of the most common methods as possible, so as Western Blots, both caspase and PARP, will likely be part of the study, but we would also like to have information using a common caspase flow cytometry assay. The idea being that by showing that Raman spectroscopic platform combined with our advanced data mining methods can non-invasively provide similar information to that obtained by performing numerous different commonly utilized assay methods, in a much more rapid manner. Thus, we would really like to test a flow-based caspase assay. So really, what I am looking for is how to improve upon what I have done thus far using this assay, and execute the assay to obtain the data in this fashion for comparison. So really it seems I need to figure out how to better improve my methodology used to execute this particular assay.

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Answer added to:
3 How quantify GSH via a Monochlorobimane protocol?
By Raphael Vidal · Universidade Federal do Rio de Janeiro

Gina Song · Seoul National University

thanx, I will read the article for experiment!

thanx, I will read the article for experiment!

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23 What is the best way to separate human granulocytes from whole blood or buffy-coat? How long can neutrophils survive in culture media?
By Rocco Cantisani · Novartis Vaccines

Silvia Caceres · National Jewish Health

I used polymorphoprep several times and went I did flow analysis, the cells were very auto fluorescence (compare to my normal percoll gradient separat... [more]

I used polymorphoprep several times and went I did flow analysis, the cells were very auto fluorescence (compare to my normal percoll gradient separation).My lab has been using the same protocol for over 20 years now and works great. It's basically a dextran sedimentation followed by a percoll gradient. Works great with big and small amount of blood. Would be happy to share some papers showing the protocol.

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14 Could FACS be modified to sort on the basis of radioactivity rather than fluorescence?
By Timothy Mclerran · University of New England (USA)

Timothy Mclerran · University of New England (USA)

Just to steer the discussion a bit: I am grateful to those who have suggested that I use conventional fluorescence and side-scatter methods to sort ... [more]

Just to steer the discussion a bit: I am grateful to those who have suggested that I use conventional fluorescence and side-scatter methods to sort cells, followed by scintillation counting to measure the radioactivity of the subdivided cell populations. This approach makes sense, and would provide the desired data for many experiments without requiring the use of RACS (thank you Dr. Mahfouz for the term). I should clarify that I am interested in the application of technology in a new way that will allow new experiments and procedures. If RACS can be done with existing technology (which seems likely, considering Dr. Holland's comment), then I believe it is worth the effort to build.

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8 Can I use both cells and beads for the compensation?
By Paola Martinez Murillo · Karolinska Institute

Paola Martinez Murillo · Karolinska Institute

Thank you very much for take the time and answer me...Finally, I used cells and always have added the FMO controls. That is extremely important.

Thank you very much for take the time and answer me...Finally, I used cells and always have added the FMO controls. That is extremely important.

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6 What are the best markers for the identification of Neutrophils, eosinophils and circulating dendritic cells in whole blood by flow cytometry?
By Massimo Caruso · Università degli Studi di Catania

Christina Stoeckle · Universität Bern

Neutrophils and eosinophils are both in the granulocyte gate, neutrophils are CD16 positive and eosinophils are CD16 negative. CD15 is expressed both ... [more]

Neutrophils and eosinophils are both in the granulocyte gate, neutrophils are CD16 positive and eosinophils are CD16 negative. CD15 is expressed both on eosinophils and neutrophils. You can even distinguish eosinophils in unstained samples by their autofluorescence

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3 Does anyone have a protocol to measure ROS formation in Saccharomyces cerevisiae with flow cytometry ?
By Philippe Marullo · Université Victor Segalen Bordeaux 2

Jan Gielis · Universitair Ziekenhuis Antwerpen

DMSO freezes easily on ice, even at 4°C it becomes solid. Perhaps the temperature on which you incubate it can be varied?

DMSO freezes easily on ice, even at 4°C it becomes solid. Perhaps the temperature on which you incubate it can be varied?

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7 Can anyone recommend an antibody that stains mouse astrocytes and works in flow cytometry?
By Alexander Scholz · Goethe-Universität Frankfurt am Main

Stefanie Lang · Friedrich-Alexander Universität Erlangen-Nürnberg

Hi everyone, I just read this thread with huge interest. Can someone recommend a good protocol for doing FACS quantifications of murine microglia cel... [more]

Hi everyone, I just read this thread with huge interest. Can someone recommend a good protocol for doing FACS quantifications of murine microglia cells? I would like to stain with CD45 and CD11b. But during isolation from brain tissue I either have still too much myelin in my cell suspension or lost too many cells by the myelin removal... Any advice or protocols? My protocol includes an enzymatic digestion step with trypsin and a 30% percoll gradient step... Thanks for your answers!

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16 I would like to know if somebody has already worked with a homemade or commercial internal control or calibrator in flow cytometry experimentation?
By Romuald Mentaverri · Université de Picardie Jules Verne

Daniele Chaves-Moreira · Johns Hopkins Medicine

Hi Romuald, Yes, I did a calibration in flow cytometer. The signals are possible to check using microbeads, and there are many types of beads availa... [more]

Hi Romuald, Yes, I did a calibration in flow cytometer. The signals are possible to check using microbeads, and there are many types of beads available. You can find some advices in these articles: 1.Sutherland DR, Marsh JCW, Davison J et al. Differential sensitivity of CD34 epitopes to cleavage by Pasteurella haemolytica glycoprotease: implications for purification of CD34-positive progenitor cells. Exp Hematol 1992; 20:590-9. 2. Schwartz A, Marti GE, Poon R, Gratama JW, Fernández-Repollet E. Standardizing flow cytometry: a classification system of fluorescence standards used for flow cytometry Cytometry 1998; 33:106-14. 3. Kraan J, Gratama JW, Keeney M, D’Hautcourt JL. Setting up and calibration of a flow cytometer for multicolour immunophenotyping. J Biol Regul Homeost Agents 2003; 17: 223-33. Good luck

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About Flow Cytometry

Technique using an instrument system for making, processing, and displaying one or more measurements on individual cells obtained from a cell suspension. Cells are usually stained with one or more fluorescent dyes specific to cell components of interest, e.g., DNA, and fluorescence of each cell is measured as it rapidly transverses the excitation beam (laser or mercury arc lamp). Fluorescence provides a quantitative measure of various biochemical and biophysical properties of the cell, as well as a basis for cell sorting. Other measurable optical parameters include light absorption and light scattering, the latter being applicable to the measurement of cell size, shape, density, granularity, and stain uptake.

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Flow Cytometry

About Flow Cytometry

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