At 3:20 PM +0100 11/2/00, tessa kerre wrote: >Dear flowers, > >In addition to Dr. Waddick's questions concerning CFSE, we too have some >problems with it. > >I have been using CFSE to track human cord blood HSC in vivo and check >for cell divisions. > >- I cannot use frozen material, because then I get clump forming, which >results in loss of 75% of my cells. Is this normal? >- It is extremely difficult to compensate the overflow from CFSE towards >the FL-3 and the FL-2 channel, making it difficult top combine CFSE with >PE or Cy labeled antibodies. Gating out of dead cells using propidium >iodide also becomes very difficult. Do others also have the same >problems, or do you have the magical settings? You can try several things... - Use a bright FL2 stain and reduce FL2 detector amplification (We actually changed suppliers to get one bright enough) - Reduce the CFSE concentration (esp. if your are analysing less than 24 hours after labelling). This will reduce the intensity of the initial staining but may allow you to compensate correctly (this might also help you with your clumping problem) - Compensation samples require large numbers of cells to allow you enough time to fiddle, especially the first few times (after that we find that our settings are reasonably stable) >- How long can you keep CFSE in good shape frozen in DMSO (-20°C)? > We have kept stocks in the -20°C freezer for well over a year. We store it in small aliquots (ie we don't ever freeze/thaw the same stock more than once, although I'm sure you could do it a few times). We also store it in a dessicator. We have noticed a slight degradation in staining intensity over that period - you don't really notice it until you make up a new batch and see how much brighter it is. Late last year we switched to storing it at -70°C so we will see if that improves things. Adrian ****************************************************** Adrian Smith (PhD Student) T CELL BIOLOGY GROUP Centenary Institute of Cancer Medicine & Cell Biology Locked Bag No.6 Newtown, NSW 2042 AUSTRALIA. ******************************************************
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