Whilst hypotonic lyse can give you absolute brilliant pictures for normal blood you run in deep trouble with abnormal samples. You tend to loose for example a lot of blasts. It is used in some haematology analysers but under extremely well controlled temperature and timings with membrane stabilising agents added. The original facslyse was meant to make allow measurement of lysed cells using the old FACS analyser that used a Coulter volume orifice. A bit like shrinking the cells down to their nuclei with the good old Coulter counter. As everyone should do from time to time, have a look down the microscope to see the alteration in the cells morphology. Any form of lyse or separation introduces artefacts, as does the process of taking the samples in the first place. Thus the best method would be to measure w/o lysis in order to avoid artefacts. There are even attempts under way to make intracellular stains w/o lysing and fixing. The ultimate dream might be something like the "Cyto Sub" or a "Guava PC" equivalent at nano-scale to be injected into your body with a little anchor. Regards Gerhard -----Original Message----- From: Akpinar, Edip (NIDDK) [SMTP:EdipA@intra.niddk.nih.gov] Sent: Wednesday, August 22, 2001 12:25 PM To: Cytometry Mailing List Subject: RE: whole blood lysis and scatter plots 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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