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Staining of Intracellular Cytokines


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#1 borriello.87

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Posted 27 August 2009 - 04:34 PM

Hi all,
I'm going to measure cytokine production by human lymphocyte stimulated with PMA/Iono in the presence of Bleferdin A via flow cytometry and I have some questions for you.
Do I have to block Fc Receptors before fixing and permeabilizing cells? How can I do this?
What is the lowest number of cell I can start with?

Thank you in advance.

#2 miBunny

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Posted 28 August 2009 - 04:54 PM

You will need to gate on CD4 and CD8. From those populations, you will need to have enough cells to generate your intracellular cytokine plots. It takes at least 5 thousand events to make these plots look good (and more is better). You will lose cells from the PMA/IONO treatment and during the wash and stain steps. So, in summation, you lose cells at multiple steps and you will be looking at subpopulations so you will need enough cells at the beginning so you can get good results at the end.

If you can start with a million cells, you should be in good shape. You can use 500,000 cells and still get ok results.

#3 miBunny

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Posted 28 August 2009 - 04:55 PM

Add Fc block after you have collected your cells and before you add your staining antibodies. Usually around 5 minutes on ice. Then proceed with your stain.

#4 borriello.87

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Posted 28 August 2009 - 11:17 PM

Thank you miBunny, your answers are always useful!
I will start from CD4+CD161+ T lymphocytes, so I won't need to gate on CD4+CD8+.
I was thinking about performing these experiments in 96-well plates, but as far as I know the maximum number of cells that can be loaded into such wells is 2*10^5 - 10^5.
So this number would be too low, right?

#5 miBunny

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Posted 04 September 2009 - 05:40 PM

It make work.... what percent of the cells will be CD4+CD161+?

#6 borriello.87

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Posted 08 September 2009 - 04:09 PM

CD3+CD4+CD161+ cells represent about 8% of PBMC.... but I would like you to answer another question.
Could you explain me in greater details how to block Fc Receptor on humsn lymphlcytes

#7 miBunny

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Posted 08 September 2009 - 06:53 PM

I simply incubate the cells with FC block for 5 minutes on ice before I add my staining antibodies. I don't wash between steps.

For mouse cells, BD has a good product. For human cells, there is a few out there.

http://www.ebioscien..._14/14-9161.htm
http://www.biolegend...tibody-568.html




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