Dear Flowers and flowerettes Thank you very very much to everybody that replied to my question about wide peaks in CFSE stained cells. The resolution improved a lot when I gated on small cells only, as suggested by one of the answer. A summary follow for those interested. Have a nice day, Mara ****************************************************************** I use a lot CFSE, and it works fine for B or T cells. I load only 5 min in RPMI with no additions, and stop with cold full medium. I use 5 uM, others use even half of that. If you try to load in PBS, you may get a too strong staining, and for sure 20 min is too long. Peaks tend to be not uniform if the cells are not of the same size. So if you have a mixture of big and small cells it may give you this phenotype. Did you try to gate on a small population in the forward versus side? best regards, For mouse leukocytes or protozoan parasites I am working with, I use 1.25 micromolar final concentration of CFSE in PBS, room temperature 5 minutes. Cells are washed twice in PBS and once in culture media thereafter. It's worked every single time for me. I do not know why you need 20-minute incubation, as it is said a concentration greater than 2.5 uM of CFSE can be toxic to cells, particularly when incubation time is prolonged. Another thing I would try to avoid is to prepare CFSE stock in serum-containing solution, which will reduce staining efficiency. I would recommend re- evaluating your CFSE staining conditions. It sounds like the concentration of CFSE is too low - that is when we get wide peaks. We do a matrix of CFSE concentration and time labeling to fine tune for each cell type, then look at the width of the labeled cell peak in FL1 to select optimal conditions. It is most important to make sure that there are no primary amine groups in the medium while you are labeling and that excess protein has been washed off the surface of the cells. A N- hydroxysuccinimide ester will bond with any primary amine group which reduces the amount that labels the proteins on the inside of the cell. However, you need protein in the medium you resuspend in after labeling to protect cells while they repair membranes. I don't know how much time is passing between staining and analysis, but CV's can improve for dyes that require ester cleavage (succinimide and acetoxymethyl esters, for example) with increased incubation time before or after staining. In my hands, after 60' of Indo'1 AM staining, max signal is attained about 30' AFTER the initial 60' staining period. The other thing to consider is your CFSE stock. If it sat around for 8 months, perhaps it's lost some of its "zip". "The information contained in this email is confidential. It may also be legally privileged. It is intended only for the stated addressee(s) and access to it by any other person is unauthorised. If you are not an addressee, you must not disclose, copy, circulate or in any other way use or rely on the information contained in this email. Such unauthorised use may be unlawful. If you have received this email in error please reply to it immediately and delete it and all copies from your system."
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