>Hi, I thought that since there was so much interest in CFSE I would >share my recent experiences. I hope it will be of some use. > >I have been labelling human PBMC with CFSE according to Current >Protocols methodology. After labelling, my cells were more than 95% >viable by trypan blue exclusion... but would not proliferate in culture. >In the end, by titration, I found that reducing the final concentration >of CFSE to 2 micromolar, (instead of the suggested 10 micromolar) gave >good staining while still allowing good proliferation. > >So the moral of the story seems to be that CSFE can do funny things to >cells, and although they are viable they may not be fully functional! > > >-- >Lisa M Gale >Department of Microbiology & Immunology >University of Adelaide >ph +61 8 8303 4630 >fax +61 8 8303 4362 >email lisa.gale@adelaide.edu.au Yes, it always pays to be careful. People have noted effects of CFSE on cells as far back as 1994 (at least that was the earliest publication I could find)... for example... >De Clerck, L. S., et al. (1994) Use of fluorescent dyes in the >determination of adherence of human leucocytes to endothelial cells >and the effect of fluorochromes on cellular function. Journal of >Immunological Methods 172(1): 115-24. >A number of supravital fluorochromes are available to study >leucocyte functions in vitro and in vivo. The fluorescein ester most >widely used, fluorescein diacetate, has the disadvantage of rapid >cellular efflux, whereas more recently developed fluorescent probes >do not exhibit this inconvenient trait. However, their effect on >cellular functions has not been thoroughly investigated in humans. >In this study, we describe a simple and rapid fluorometric method >for measuring cell adhesion to endothelium, comparing 5 different >fluorochromes. Furthermore, we evaluated the effect of fluorescent >dye labelling (with CFDA, CFSE, BCECF-AM, calcein-AM or DiI), on >various cell functions, including, apart from adhesion, lymphocyte >proliferation, granulocyte chemotaxis and superoxide production. >calcein-AM and DiI proved to be the fluorochromes with the least >effect on cellular function. BCECF-AM did not interfere with >lymphocyte proliferation, but exhibited some influence on superoxide >production and chemotaxis of granulocytes. CFDA showed a detrimental >effect on both lymphocyte and granulocyte functions whereas CFSE >gave intermediate results. In the adhesion assay, calcein-AM, CFSE >and DiI performed comparably well. Since labelling with C12-DiI was >homogeneous, this probe was also appropriate for the adhesion test, >although somewhat higher background staining was present. We >conclude that the fluorochromes are powerful tools when analysing >the adhesion of human leucocytes to endothelial cells. However, >since fluorochrome labelling can interfere with other cellular >functions, the fluorescent probe has to be carefully chosen with >regard to the cell type and function to be studied. (NOTE: I haven't heard of CFSE affecting murine cells in the same way. Anyone?) Adrian Smith ****************************************************** 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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