I am trying to look at proliferation and cytokine (IFN-g) production on PBMC's with and without blocking antibodies. Trying to demonstrate that there is a difference in T cell function with and without blocking.
I am starting with PBMC stim with OKT3 for 5 days. But I notice that at 5 days barely I can see any IFN on intracellular staining. What do you all suggest? These are not sorted T cells but PBMC's which are thawed after cryo preservation.
Thanks
I have done a similar experiment without a problem, so I guess it should work. Do you have a positive control for intracellular staining of IFN-g to sort out whether the problem is caused by staining or culture condition etc?
Hi Krupa,
CFSE and intracellular cytokine staining can be done simultaneously. I would avoid using a PE conjugate for your ICS stain as CFSE tends to leak into that channel a fair bit. APC for ICS along with CFSE has worked well for us in the past.
Intracellular staining is difficult to see without a Golgi complex inhibitor eg. Monensin or Brefeldin A and I am not sure if you are using them. Those compounds are toxic in the long term so you don't want to keep them much longer than 6-8 hours. You could try your 5 day stim with OKT3 and then restimulate for 6 hours in the presence of a golgi inhibitor prior to doing ICS.
Good luck!
Hi Luis, thanks for answeing. We use Golgi plug and stop 6 hits prior. So, should I go ahead and add restim with PMA/Iono at the same time??
Thanks
Do you stimulate Antigen specific ? Could you stain the CFSE cells with Tetramers ? In General maybe you need to culture the cells like 7 days...
http://jem.rupress.org/content/early/2012/01/03/jem.20100388.1.short
Hi Krupa,
If I understand correctly, you are adding Golgi plug at the last six hours of your 5 day incubation? If so, I think restimulation (PMA/Iono or OKT3 on a fresh plate) for six hours with Golgi plug would work better.
Antigen specific stimulations will have a smaller responder population so maybe not something I'd dive into until you have troubleshot your culture conditions. However, Julian's idea of a 7 day culture may work since you are starting with frozen PBMCs. I have seen labs have terrible results with experiments due to inadequate freezing of PBMCs so be sure that your thawed cells show decent viability if the restim does not work.
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