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questions about PBMCs study - (Jul/22/2013 )

Hi, I want to analyze if a stimulant could stimulate immune response from patients' peripheral blood mononuclear cells (PBMCs). I have some questions here:

1. How should I testify if the immune response is activated? What parameters should I choose?

2. I see papers test cytokines. So I want to ask how should I choose which cytokines to test?

3. Besides cytoknies, some publications also include Cell proliferation assay. I tried BrdU kit but it doesn't work well. Is cell proliferation assay necessary?

I look forward for your suggestions. THANKS VERY MUCH!

-joy123-

(Warning - I'm not an immunologist, so may give not such good advice here)

Yes, cytokines (e.g. interleukins) would be appropriate to measure, as would T-cell or B-cell activation.

How to choose depends on what you are looking at - bacteria will produce a different response to viruses etc.

Cell proliferation could be a marker, I would go with MTT/MTS assays or a simple cell count (maybe by Flow cytometry) rather than BrdU.

-bob1-

bob1 on Tue Jul 23 10:01:08 2013 said:


(Warning - I'm not an immunologist, so may give not such good advice here)

Yes, cytokines (e.g. interleukins) would be appropriate to measure, as would T-cell or B-cell activation.

How to choose depends on what you are looking at - bacteria will produce a different response to viruses etc.

Cell proliferation could be a marker, I would go with MTT/MTS assays or a simple cell count (maybe by Flow cytometry) rather than BrdU.


Thanks! But cell proliferation is not quite realistic for me. Can I skip it? If so, should I do something else to supplement the cytokine analysis?

-joy123-

No idea, sorry. There is bound to be something else that can be done (migration assays perhaps?).

-bob1-

Hello:
If you want to get the percentage of activated cells you have to know that markers used, for example CD25 and / or CD69 in T cells.
The proliferation assay is also a basic tool: BrdU could be good, I usually use CFSE.
And finally if you want to evaluate cytokines, you have to know what cytokines are involved in the process that you are evaluating.
 
Good Luck.

-waldomedina-