From: Dr. Michael G. Ormerod (ormerod@icr.ac.uk)
Date: Thu Dec 19 1996 - 10:38:02 EST
Thanks for responding. The reason for using FACSlyse was that we wanted to lyse the red cells and to fix the white cells in paraformaldehyde. The paraformaldehyde fixation stops low MW fragments of DNA diffusing out of the apoptotic cells. FACSlyse offered a convenient one step procedure for doing this. Michael Ormerod On 12 Dec 1996 10:35:27 Z Gerhard Nebe-von-Caron <Gerhard.Nebe-von-Caron@unilever.com> wrote: > > Hi Mike > > I always observe the same with Facslyse and PI (so not doing > detailed DNA analysis. Why do you want to use facslyse? It > is a fixative lyse made up these days from a mixture of at > least formaldehyde and minute quantities of glutaraldehyde. > Have you tried the old dist-water lyse? 50ulblood+ > 1800uldist water for 15 to 20 seconds followed by 150ul > 10xPBS. > > Good luck, peacefull Christmas and a healthy 1997 > Gerhard Nebe-v.Caron > Unilever Research, Colworth, > Sharnbrook, Bedfordshire > GB - MK44 1LQ > Tel: +44(0)1234-222066 > FAX: +44(0)1234-222344 > gerhard.nebe-von-caron@unilever.com > > > ______________________________ Reply Separator _________________________________ > Subject: PI staining of PBLs > Author: ormerod@icr.ac.uk at INTERNET > Date: 11/12/96 04:16 > > > I have run into a probelm staining peripheral blood cells > with propidium iodide for cell cycle analysis. We took > whole blood and lysed the red cells with FACSlyse. The > cells were then washed and fixed in 70% ethanol on ice. > After 1 to 24 hours, the cells were washed and resuspended > in a buffer containing PI and RNase and incubated at 37 for > 30 min. We have used a variety of different buffers. > > We consitently obtain two peaks in the DNA histogram. A > lower peak from the lymphocytes and a peak from the > granulocytes with about 30% more fluorescence. > > If we use a detergent method to prepare unfixed nuclei, > there is only one peak in the DNA histogram. > > The only reference I can find to a differential staining of > lymphocytes and granulocytes is by Stokke and Steen > (Cytometry 8, 576-583, 1987) who observed a difference > when they stained formaldehyde fixed PBLs with 7-AAD. > > Has anyone else observed this effect? Does anyone know how > to persuade the the two types of cell to take up the same > amount of PI? > > The reason for this esperiment is that I want to fix bloody > tumour samples for a Tdt assay and I do not want to be > confused by two peaks from the normal cells. > > Michael Ormerod > preferred email address: 100537.2462@compuserve.com
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