I want to observe IL10 secretion by lymphocytic cells, could anyone of you guys suggest to me how much splenocytic/lymphocytic cell I need to plate for 6 well/12 well/24 well plates
You should be able to harvest large numbers of splenocytes from a single mouse spleen, so I would imagine you could use anywhere from 0.5 - 1*10^6 splenocytes/ml in a 6 well, 12 well, or 24 well plate.
In a 24 well plate we did 1x10^6 as standard - and from a spleen you could get around 30m splenocytes. We would then culture them for a week in either Th1 or Th1 polarising conditions (IL-12 with IL-2 d3 and 5 or IL-4 and anti-IFNy with IL-2 d 3 and 5) then restimulate with either irradiated splenocytes and antigen, or anti CD3 and 28 beads/coated plates
at sounds like you plan on using a heterogenous population of cells but than ant B-cell specific IL-10 secretion. In the current set-up it is hard to be sure it is B-cell specific. The Th polarization as suggested earlier by Charly Oakley doesn't solve this either.
If you have the possibility, you could go for intracellullar IL-10 staining in combination with eg CD3 to exclude T cells, and CD19 to select for B cells. CD19 covers most B cell subsets.
Thanks, for your valuable information, with FACS analysis and CD19 I want to see specifically the B cell which secrets IL10, also I am isolating specificall B cell with Stem cell technologies B cell isolation kit and like to find out only B cell population could secret IL10 or not
Gerco here is absolutely right. if you are purifying B cells with a kit (preferably "untouched" B cells) you should be OK. Just make sure you have different stimuli (CD40, LPS, BCR) and that you actually look at intracellular cytokine production at the right time-points. Alternatively, I would also suggest that you collect the supernatant, where you can always run an ELISA for IL-10. Good luck!
Yah, thats what my plan, after isolating only B cell I like to use different stimuli (LPS+PMA+INONOMYCIN), CD40, BCRparasitic antigen to have an information regarding IL10 secretion from B cell by running ELISA
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