Fixation and (PE) fluorescence

From: Gerhard Nebe-von-Caron (Gerhard.Nebe-von-Caron@unilever.com)
Date: Wed Jul 02 1997 - 12:58:22 EST


          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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