Hello, I am trying developing a culture system using human platelet rich plasma and rat osteoprogenitor cells. I am forming clots out of the PRP and then adding the rat bone marrow cells. I am interested in the response of the rat bone marrow cells and bone development. As you can imagine there are many different cells types in the culture (rat BM cells, human platelets, leukocytes, red blood cells, etc). I have labelled the rat cells with CFSE (molecular probes) prior to introduction to the PRP clots, and I'm having some difficulty with performing the flow cytometry (FACSCalibur). The dot plots that are produced are all over the place because of the different sizes of all the cells present and also rat osteoblasts fall in the size range of human leukocytes. The peaks on the histogram are also not well defined. I was hoping that someone maybe able to provide me with some insight into settings and tips when using flow with CFSE. As you may well imagine I am dealing with a lot of auto-fluorescence from the red cells. The settings that I am using are: Detector Voltage AmpGain Mode FSC E-1 7.00 Lin SSC 377 1.00 Lin FL1 551 1.00 Log I have not fixed any of the samples before reading on the flow cytometer, as it may cause higher auto-fluorescence. I would appreciate any insight or tips anyone might have regarding examining only a CFSE labelled population in a mixed culture using flow cytometry. Thank You, Chris Rose BSc, CTBS, MASc (c) School of Biomedical Engineering Dalhousie University
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