Re: RE: PI staining of PBLs

Dr. Michael G. Ormerod (ormerod@icr.ac.uk)
Thu, 19 Dec 1996 10:35:23 -0500 (EST)

Thank you for your reply. The temperature of incubation was
immaterial. The separation of the two peaks increased with time of
incubation, the lower temperature just slowed things down. The
reason for incubatin was to remove double stranded RNA, not so
important for leucocytes but more important for tumour cells.

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
> >
> >
> >