Greetings flowers, I am preparing to start some work looking at intracellular cytokines in mouse blood and have a few questions. Does anyone know the reference ranges for lymphocyte subsets in mouse blood? We may only be able to work with about 0.25 ml of blood. Has anyone had success staining for cytokines with this small of an amount? Also, the blood will be drawn at a different facility, stimulated with PMA+ionomycin in the presence of brefeldinA, and then must be fixed for safety reasons. Samples will then be delivered to us (about 2-4 hours post-fixation) to be permeablilzed, stained and analyzed. What are your opinions on how long the fixed cells could be stored prior to analysis assuming we used fresh high-quality formaldehyde? I know that fixation can wreak havoc on some surface proteins. Does anyone have experience staining with anti-CD4 (clone RM4-5, BD biosciences) prior to or following fixation? Perhaps someone out there doing similar work wouldn't mind sharing their protocol with me? Thanks. Jake Kohlmeier Department of Molecular Biosciences University of Kansas 1200 Sunnyside Ave. 7035 Haworth Hall Lawrence, KS 66045 Ph (785) 864-4029 Fx (785) 864-5294
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