Intracellular Cytokine - Optimal time

RAJA ALAMELU alameluraja at yahoo.com
Thu Jun 10 05:22:44 EST 2004


Dear FLOWers,
In my laboratory, I am setting up Intracellular cytokine (IFN-g, IL-2, TNF-a and IL-4)
assays using FACS Calibur, in normal human and tuberculosis peripheral blood mononuclear
cells. I stimulate the cells with M. tuberculosis purified antigens, incubate for 72 hrs
at the end of which I add Brefeldin to the cultures. After an additional 16 hrs of
incubation with Brefeldin, I process the cells for intracellular cytokine (ICC)
measurement, using antibodies from B-D.
1.   Can anyone using microbial antigens for stimulation let me know, what is the optimal
time of incubation with antigens for ICC? Is it the same for all the 4 cytokines?
2.   What is the optimal time of incubation with Brefeldin (16 hrs is practically
convenient)?
3.   After the in vitro cell culture for nearly 96 hrs, I find a lot of dead cells in the
lymphocyte scatter gate, which form autofluorescent signals in the double-positive
quadrant. Is there any way to avoid them?
4.  I included CD45 FITC in all the tubes. On an SSC vs CD45 plot 2 bunches of
lymphocytes clearly separate, of which the higher intensity correspond to the live cells.
I can analyse the ICC within this gate in all the tubes. However, CD45 is a very
expensive reagent to be included in each and every tube. Is there any other alternative?
Thanking you in anticipation,
With regards,
Alamelu Raja   


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