6 replies to this topic

[zodiac1505]

I have been trying to do T helper differentiation and to stain for intracellular cytokines. I think the differentiation works fine since I can see loads of cytokines by ELISA but I can never see anything if I stain the cells and do FACS. I used the staining kit from BD and antibodies from R&D and eBioscience. I usually restimulate the cells with PMA+ ionomycin. I have tried both golgiplug and brefeldin A. Does anyone have any idea what could be wrong?

[Piersgb]

Which detergent are you using and how much of it? It could be that the detergent that you are using is too strong and is destroying the cells? Have you done any fixation steps?

[zodiac1505]

Piersgb, on 13 September 2010 - 02:26 AM, said:

Which detergent are you using and how much of it? It could be that the detergent that you are using is too strong and is destroying the cells? Have you done any fixation steps?

I am using the BD intracellular cytokine staining kit. It contains the fix/ perm solution.

[Denis Baev]

Before IC staining for cytokines you HAVE TO stimulate your cells (to boost the cytokine production up) by TCR stimulation (anti-CD3/anti-CD28 antibodies) or by PMA/Ionomycin for 4-6 hrs. Dont forget to block the sytoken secretion by brefeldine after 3 hrs of stimulation (the "how to...." step-by-step instruction have to be in your BD IC staining kit manual book)
If you did everything corretly your staining have to be OK (i never had prolems with BD and eBioscience kits).

Edited by Denis Baev, 25 January 2011 - 06:02 AM.

[zodiac1505]

Denis Baev, on 25 January 2011 - 06:00 AM, said:

Before IC staining for cytokines you HAVE TO stimulate your cells (to boost the cytokine production up) by TCR stimulation (anti-CD3/anti-CD28 antibodies) or by PMA/Ionomycin for 4-6 hrs. Dont forget to block the sytoken secretion by brefeldine after 3 hrs of stimulation (the "how to...." step-by-step instruction have to be in your BD IC staining kit manual book)
If you did everything corretly your staining have to be OK (i never had prolems with BD and eBioscience kits).

I usually restimulate the cells with PMA/ iono with Brefeldin before staining. After I posted this, that staining worked for me quite well for 2 times and then its again back to nothing. I am going to try again with newly ordered cytokines and see what happens. Keeping my fingers crossed.

[Denis Baev]

By the way how did you store Bref and PMA? These reagents have to be stored in freezer but don't like repeated freezing-thawing...

[zodiac1505]

Denis Baev, on 27 January 2011 - 06:00 AM, said:

By the way how did you store Bref and PMA? These reagents have to be stored in freezer but don't like repeated freezing-thawing...

I have brefeldin in the fridge according to the recommendation (it remains frozen in fridge). I guess using it involves several freeze thaw cycles. PMA is in the freezer and I usually use aliquots which don't last for more than a couple of times. Planning to do single use aliquots from now on

Edited by zodiac1505, 27 January 2011 - 06:13 AM.