Dear flowers, Could anyone of you tell me what are the best conditions (mainly time of restimulation, temperature of incubation, etc) to detect intracellular IL-4 in splenocytes in response to mitogen or antigen restimulation? We are working with splenocytes from parasite-infected animals. In ELISA we can detect IFN-gamma and IL-4 in the supernate when these cells are restimulated in vitro with mitogens such as ConA. However, whereas we can detect IFN-gamma intracellularly by flowcytometry after overnight incubation with ConA, we do not detect IL-4. After the overnight incubation with ConA, we are treating the cells for 5 h with Brefeldin A before the FACS staining. We are using Pharmingen's R-PE-conjugates anti-mouse IL-4 antibody (clone 11B11) at 0.2 microgram/one million cells and we are staining paraformaldehyde-fixed and saponin-permeabilized cells for 30 minutes at 4°C in the dark. Thanks in advance for any advice on this matter, Geert Raes Laboratory of Cellular Immunology Free University of Brussels Belgium
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