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Michael Ormerod
On Thu, 12 Dec 1996 09:29:38 -0500 "Barren, Phil" <BarrenP@MedImmune.com> wrote:
> Response to Twin Peaks:
>
> I think your looking at some articfact staining the grans and lymophs
> will take up DNA dyes differently but the peaks should be almost
> superimposable, irregardless of the DNA dye you use.
>
> Why are you staining at 37C? Most protocols are at 25C or 4C.
> I used to work for BDIS and my experience with their DNA kit
> was always good.
>
> >----------
> >From: Dr. Michael G. Ormerod[SMTP:ormerod@icr.ac.uk]
> >Sent: Tuesday, December 10, 1996 5:36 PM
> >To: Cytometry Mailing List
> >Subject: PI staining of PBLs
> >
> >
> >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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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
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