Thankyou for this protocol. I am following almost same protocol...

I have the same questions, when I do stimulation of mouse splenocytes with 500 ng/ml ionomycin, 500 ng/ml PMA and Golgistop at the same time fro about 4 hours, the cells looked like they all die or lyse. So I have to stop at this step. Did I add too much PMA? but according to BD protocol, IL-17 stimulation need this condition. Should I add Golgistop right after stimulation?

Hi Priyanka,
Thank you for your inquiry. Before we can get any solutions, I think it is important to find out whether the cells are not activated (ineffective stimulator?) or the staining procedure is not right (ineffective fixation/permilizaiton or bad antibodies?). So please help me answer the following questions which might help us figure out what's wrong with your experiment.
1. What type of cells are you using? Is it PBMC from human blood?
2. How long did you stimulate the cells? Is there any cell-aggregation (clusters) present in the culture?
3. Did you add BD GolgiStop right after you add PMA/Ionomycin?
4. What's the signal level of CD4 staining? There are reports stating that PMA stimulation might downregulate the expression of surface CD4, so an intracelluar CD4 staining might be helpful (http://www.ncbi.nlm.nih.gov/pubmed/11506744).
5. Did you incubate the antibodies in the 1X Perm/Wash buffer?
6. Are you able to observe any fluorescent signals in your samples?
I will get back to you as soon as I get your answers.
Thanks,
Huagang