(First, I would like to thank all who responded to my question of the FACScan upgrade...we have not made a decision yet. If anything noteworthy occurs, I'll report it.) We are doing calcium flux experiments on freshly isolated PBMCs that are also labeled with 4 fluorochromes (FITC, PE, Cychrome and APC). Unlike any other cells that I run on the Vantage, the lymphocyte population has a relatively broad SSC ( although the population is well defined and isolated). In addition, what is more troubling concerns the negative populations in the fluorescence (density) plots. For example, in the FITC vs. PE plot, the negative population is so "wide" or diffuse, that , no matter how high I push the voltage, I can not see the entire negative population. When the voltage is increased, the negative pop. becomes larger and larger. This makes proper compensation practically impossible, since I can not visualize my entire negative poulation. The positive populations are much tighter. Loading of the cells with INDO-1 has been ruled out as the source of this problem. SInce I often have all 3 lasers going (488, uv, HeNe) for other experiments and do not have this problem, I don't think that the problem is due to multilaser excitation, etc. Briefly, this is what the cells do before I see them. Blood is drawn into vacutainer, followed by layering over Histopaque, centrifugation for 30 min at 800xg. The lymphs are resuspended in RPMI and washed 3 times (800xg). THey are incubated 45 min at 37oC to remove monocytes, centrifuged and resuspended and incubated in RPMI /2%FBS and 10 uM indo-1-AM for 45 minutes. They are washed and resuspended in complete RPMI with 0.2% azide at 50 million/ml. FInally, they are stained in 96 well plates, washed and resuspended in RPMI and diluted to 1 ml in HBSS in flow tubes. Has anyone had this problem? Should I just accept it if I can't resolve it? Will the experts always question the validity of the data if the whole negative population can not be seen? How critical is it? There is always the question of proper alignment, but the fact that I don't see these phenomena with any other samples does not support a basic problem with operation of the machine.. Actually, I don't ever use freshly isolated human PBMCs...is this a characteristic of human cells...? How about all of the centrifuging and resuspending? Is that changing the morphology/autofluorescence of the cells? Thank you for reading this lengthy description. I would love some comments! SIncerely, Tina Rogers TIna S. Rogers, Ph.D. Manager, MAMDC FACS Core Facility Division of Rheumatology University of Alabama at Birmingham 1900 University Blvd. THT 405 Birmingham, AL 35294-0006 205-934-1362
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