Intracellular IL2? - (Feb/14/2008 )

Does anyone have a protocol to staining intracellular IL-2 for flow cytometry analysis?

thanx :-)

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I have not doen a IL-2 staining but I think most cytokines intracellular staining share the similar protocol:

1. after your last treatment, add PMA and Inomycin to stimulate cytokine production in the presence of monesin (a Golgi inhibitor, it helps IL-2 stay within the cell) for 5 hours(add monesin the the last 2 hours).

2. Fix and permeabilize cells

3.Antibody against IL-2.

4.Go to flow cytometry.

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Stimulate cells with TPA/ionomycin, in the presence of monensin or brefeldin A to inhibit release of cytokine
stain the extracellular antigens as usual
Fix the cells with PFA 4% 20 min RT
wash
incubate with anti-IL2 in the presence of saponin, 0.1% (try several concentrations) to allow the entry of the antibody, 1 hour 4¨C.
wash twice with saponin and once without.
go to flow cytometry

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I've tryed this protocol already. I could see IFNy but no IL2.
But I also had only very few CD4+ cells. So I will try today again, I will do O/N with stop golgi.
Have you ever tryed anti IL-2-FITC from BD? They say there to use 20µL, but I have so many samples and at this
concentration I would use up the entire new IL2 tube I bought...
Do you think I could use 10µL instead of 20?
cheers

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I never used this antibody.
When you buy a new antibody, try several concentrations. I would not use 10 µL instead of 20 on all my samples, but you could just try to compare 20, 10 and maybe 5 µL on one sample.

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