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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