Dear FLOWers I'm studying T-helper cells from whole blood, but am new to flow cytometry. I've found that when I use 1XFACSlyse for 10mins (2ml added to 100ul blood for 10min ) following antigen labelling e.g CD4-FITC, there is a marked decrease in the Forward scatter of lymphocytes (such that they appear at ~100-200 on a 0-1023 scale and merge with debris) when compared with forward scatter of lymphocytes from unlysed mononuclear cells following Ficoll gradient separation-these lymphocytes appear at ~250-400 using the same cytometer settings. I'm using a FACSscan and have set up 3 colour compensation using single-fluorochrome labelled mononuclear cells after Ficoll separation. Is this a normal effect with FACSlyse, and if so do people simply increase the FSC gain to drag the lymphocytes back over, and should I then re-do the colour compensation? Sorry if this is a bit basic but I'm a bit stuck. Many thanks Francis Andrews Dept of Medicine University of Liverpool fandrews@liv.ac.uk
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