Phellow Phlowers, I am working with several adherent cell lines including the murine macrophage line J774A.1 and have had problems with very high background in the FL2 parameter with the macrophages. I am using a FACS Calibur to try and detect surface markers after trypsinizing and staining the cells. The negative isotype control is giving me a histogram peak channel of between 10 2 and 10 3 which is about a log scale higher than my positive control (murine thymocytes).When I try to reduce sensitivity by lowering FL2 voltage I lose the ability to detect the positive control. I haven't had this high background problem in my other adherent cell lines. Things I have tried to reduce the background include using Fc block from Pharmigen, blocking with rat gamma globulin, culturing the cells in RMPI w/out phenol red, and letting the cells "heal" in complete media before staining (I thought that membrane integrity might be comprimised after trypsinization leading to the uptake of flourochrome conjugates).I am using a setting of around 585 for FL2 which might be a bit hot, but any setting much lower results in a large loss of sensitivity. Any help or ideas would be greatly appreciated.Thanks. Chris Reed
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