How to improve intracellular cytokine staining for IFN-gamma in human PBMCs? Actually, I incubate cells with 50ng/ml PMA and 1ug/ml ionomycin for 5 hours. At the same time, we add Golgi stop and golgiplug but we couldn’t see any events. Is there any suggestion?
try different time points in your setting
include different concentrations
and unstimulated
this should work
Dear Thilo
Thanks for your asnswer
In this time point we detected IL-22 and IL-17, dose it make sense for IFN if change the time?
Thank you Andrea ,
We did the test with another antibody , no changes were seen in the results
For an optimal result you should first add PMA and ionomycin for 2 hours and then add Golgi stop/plus for 4 hours. Which buffer are you using for the intracellular staining?
If you use regular staining buffer you will not get any signal from intracellular cytokines. You should use specific buffers to permeabilize cells such as: http://www.bdbiosciences.com/us/applications/research/intracellular-flow/intracellular-buffers-and-ancillary-reagents/permwash-buffer/p/554723
There are a lot of alternatives out there from different companies.
I agree with Raffaello that you need to use proper permeabilisation conditions to see intracellular cytokines, but if you are already seeing other cytokines then I am guessing you're doing that.
Another question- what extracellular markers are you using in your panel, and what gating strategy are you using before looking at IFNg? The IFNg+ can be enriched as you drill down to specific subsets of PBMCs.
I also always do an FMO for IFNg since positives are pretty rare even after stimulation.
Hi Kurosh. I regularly do intracellular flow cytometry on human PBMCs and use PMA (20ng/mL) + ionomycin (1 ug/mL) as my positive control. We do 24-48 hour stimulation and add BFA+monensin for the last 4 hours of incubation. We get around 50-60% of CD4+ T cells secreting IFN-g under this condition. As has been suggested by others, gating on lymphocytes might make things easier.
I am wondering why you are able to pick IL-22 and IL-17 up but not IFN-g. IFN-g is one of the most easily/highly expressed cytokines while the frequencies of cells expected to secrete IL-22 or IL-17 are low. Have you tested whether the IL-22/IL17+ cells you see are real? Did you try using isotype controls?
We use Transcription Factor Buffer Set (BD Cat # 562725) for fixation-permeabilization and Mouse Anti-Human IFN-g Clone B27. Is there a reason for why you have a 5 hour stimulation? The shortest I have ever stimulated for is 6 hours. From my experience, TNF secretion goes down after 6 hours. Most cytokines including IFN-g go up from 6 hours to 24 hours. But it is definitely a good idea to do a kinetic study since your experimental setup might be different.
Dear all scientist,
We found the problem.
I check the CD4, Il-22.Il-17 and IFN-gamma.
The clone which we use for CD4 could detect internalized CD4 , as you all know when the cell become active some CD4 internalized, then you can bypass the surface staining.
After 5 hour stimulation we must fixed the cell then perm and add the Ab .
After washing run the cells.
The step we missed was the fixation, after doing that thing become great.
Thanks to all .
Best
Kurosh
stain for cd3 cd4 cd8 cd69
the fraction cd8+cd4-cd3+will be ifngamma +
dendant upon stimulation
add isotype control for ifn gamma antibody
and compare unstimulated
Now that you know that the buffer was your problem (fixation), you should make sure that you get a good signal for IL-17 and IL-22 (which is more difficult than IFN-g). Please titrate your antibodies, use FMOs as control and don't use isotype controls. Isotype controls are not a reliable way to define your background level of fluorescence.
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