Francis, Edip, et. al...... I believe the formaldehyde fixative in FACSLyse causes the cytoplasm of the cell to collapse around the nucleus, much like you see on a commercial hematology analyzer, while maintaining the integrity of the surface bound antibodies. This allows better separation of your major leukocyte populations on the basis of scatter. Kb -- Keith Bahjat, PhD Applications Engineering Manager Cytomation, Inc Fort Collins, CO (970) 226-2200 x223 keithb@cytomation.com on 8/22/01 5:24 AM, Akpinar, Edip (NIDDK) at EdipA@intra.niddk.nih.gov wrote: > > > 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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