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