Fellow Flowers- We are doing intracellular cytokine staining for IFNg (after PMA/Iono/Monensin) and are getting lovely results. Our problem is as follows: when we do single color staining for IFNg, we get about 40% positive. When we do a 4 color staining (CD4 FITC, CD8 PerCP, CD56 PE, and IFNg APC) without incubating with labeled anti-CD4 and anti-CD8 during the stimulation portion of the experiment (which we usually do, to address the problem of marked decreases in CD4 and CD8 staining due to internalization of these markers during stimulation), we still get about 40% staining with IFNg. However, when we *do* add labeled CD4 and CD8 antibodies during the incubation (the remainder of the 4 color staining with CD4 FITC, CD8 PerCP, CD56 PE and IFNg APC remaining unchanged), the IFNg staining is significantly reduced (to 25-30%) . Has anyone else had experience with this kind of interference and, if so, what causes it and how did you solve it? Could anti-CD4 or anti-CD8 antibodies prevent anti-IFNg antibody binding? Can quenching be occurring? or is there some other explanation? Thank you in advance for any thoughts on the matter- Regina Regina H. Harley, M.S. Lab Supervisor - Flow Cytometry Facility Center for Vaccine Development University of Maryland - Baltimore 685 West Baltimore Street Baltimore, MD 21201 (410) 706-7376 FAX: (410) 706-6205 rharley@medicine.umaryland.edu
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