Fixation and (PE) fluorescence

Gerhard Nebe-von-Caron (Gerhard.Nebe-von-Caron@unilever.com)
02 Jul 1997 18:58:22 +0100

Thanks Tom Frey and Keith Bahjat for your replies. By lack
of further comments I take it the field has not been
investigated too thoroughly. As I said, I am not an expert
in clinical FCM, so when comparing the lysing solutions I
left some on for longer than others, as non wash suggested
to me the samples could stay in the lysing solution. As I
prepared the Utilyse last, it had the shortest incubation
time of the fixative and thus the lowest quenching of the
PE. Being a two step lyse, they might well stop the
fixation with the second solution, thus no further decrease
of PE might occur, but I haven't tried long term storage in
lysing solutions for the various lysing solutions. For other
non wash lyse users it might be worthwhile adding TRIS to
raise the pH of their buffers and to stop the formaldehyde
with the free amines.

The second interesting observation that was triggert by
Keith's comment that CD38 is not effected by the fixation,
but cd 4 is. As I do not have cd38, I have tried 4,8,14,19
and 45ro, and apart from 19 they all go down to various
degrees. When keeping a sample stained with CD3*FITC,
CD8,14,18*PE and CD4PC5 in facslyse for 10, 30 and 60
minutes at 20oC , the following median changes occurred:
cd3 165, 158,154
cd14 82, 65, 59
CD8 72, 69, 65
cd19 13,12.7,13.
cd4 48,46,44
The 10 minute values correspond well to unlysed mfi's which
is some reassurance.

It looks as if the effect is not restricted to PE alone. It
would be nice if someone could come up with some ideas,
perhaps try it on beads or in a spectrofluorimeter. Perhaps
the CD19 expression is to low as to see the signal
decrease???

Thanks in advance for any comments

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

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