Hi, Kenichi, A couple of suggestions: 1. As has been suggested, centrifugation may be the key. You have fixed your cells in MEOH and they are much lighter than before fixation. I will demonstrate this to students when they are careless with this step--look in the supernatant and see how many still are there. When we sort live cells stained with Hoechst 33342, we have no trouble recovering the number indicated on the cytometer. 2. Are your proteins identifiable with monoclonal antibodies that would be appropriate for flow? Looking at cell cycle and protein expression (cyclins in particular and see Chow, et al for measuring phosphorylated proteins in Clinical Cytometry) is well described in Current Protocols in Cytometry and would require no sorting. We have found that trying MOABs that may not have been used for flow actually work there. Good luck, Kathy -- -------------------------------------------------------------- Kathy Schell Supervisor, UWCCC Flow Cytometry Facility 600 Highland Ave. K4/535 Madison, WI 53792 Voice: 608-263-0313 e-mail: kschell@facstaff.wisc.edu
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