Dear Francis: How do you explain this effect? Can this effect change any other property of cells and lead do invalid results? Should ve avoid using this kind of lysing buffers? Indeed, currently I am using hypotonic lysis, with simple water, but I am not sure if this is better than other methods. Edip AKPINAR NIH-NIDDK -----Original Message----- From: Simon Monard [mailto:smonard@trudeauinstitute.org] Sent: Tuesday, August 21, 2001 9:16 AM To: cyto-inbox Subject: Re: whole blood lysis and scatter plots Francis You always see that effect with FACS lyse, just whack up the FSC (double it) and you should get just as good lymphocyte/debris discrimination as with Ficoll separated cells. You should not need to change any compensation settings. Simon Monard FACS Lab Manager Trudeau Institute Saranac Lake NY12983 Ph 518 891 3080 X352 >>> "franis, alex and thomas andrews" <aanw38391@cableinet.co.uk> - 8/19/01 6:44 PM >>> 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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