Julie_Wieseler-Frank/CAS/UNO/UNEBR@unomail.unomaha.edu wrote: > > > I am trying to measure intracellular TNF-alpha in rat splenocytes -- the > operative word is trying. I am stimulating with LPS -- I have tried stimulation > times of 30 min to 13 hours; I have used monensin and brefeldin; i have used > plastic flasks (scraped prior to staining cells) and I have tried siliconized > tubes. When I look at cultures under the microscope, the morphology of the > cells indicates activation. > > Any insights? I would appreciate ANYTHING!! > Maybe some of these suggestions will help you: 1] Pharmingen sells prestimulated, fixed control cells. You could test your antibody, permeabilization, etc by using these as a positive control. 2] PMA/ionomycin is typically used to elicit a maximal cytokine response, try this instead of LPS and you should definitely see some responding cells. 3] Check your viability, the secretion inhibitors can be toxic. 4] Make sure you don't have EDTA in the medium, I believe this is a Ca+ dependent process. 5] Test your permeabilizton step by labeling with a McAb for some intracellural Ag. 6] Once you find some responding cells, you should probably repeat your time course to determine the optimal time to neasure TNF. 7] If you have access, try human peripheral blood mononuclaer cells. there are many references documenting the intracellular cytokine assay in this system. Good luck, Tom ***************************************************************************** Thomas W. Mc Closkey, Ph. D. Director of Flow Cytometry, North Shore University Hospital Assistant Professor of Pediatrics, New York University School of Medicine Boas Marks Biomedical Research Center, 350 Community Drive Manhasset, Long Island, New York 11030 ph: 516-562-4844 [office], 516-562-1135/4641 [lab] fax: 516-562-2866 *****************************************************************************
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