Dear Jens,
Normally IL-4 is too low to detect in PBMCs by ELISA no matter how you
stimulate the cells. However, there are some tricks that might help.
Costimulate with PMA (10ug/ml) and ionomycin (1ug/ml) or PHA and
ionomycin and try 24 48 and 72 hrs. I have also found anti CD3 (1ug/ml)
plus anti CD28 works (10ug/ml). Also include a blocking IL-4 receptor
antibody (approx 1-10 ug/ml), this prevents any IL-4 thats being made
being used by the cells and makes an enormous difference.
I also found it very difficult to detect IL-4 by intracellular staining
on normal PBMCs since the frequency of TH2 type cells in PBMCs is very
low. I only ever found IL-4 in certain types of immunodeficient patients.
Alternatively you could differentiate the T cells into TH2 cells. One
(of many methods) is by growing PBMCs in aCD3 and aCD28 plus IL-2 (5u/ml)
for 5 days then restimulating with aCD3 for 24 hrs. Then you get plently
of IL-4.
good luck
david
In article <8bFA5.281$qU.2433 at news.get2net.dk>,
"Jens Rainer Hansen" <jens.rainer at leo.dk> wrote:
> I am going to set up an IL4 assay on human lymphocytes (PBMC). Does anybody
> know what to stimulate with (PHA, PMA etc), how long, conc. etc. to get some
> IL4 in the supernatant ?
>> Jens Rainer
>>
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