Hi, I'm looking for a method to label two cell lines differentially so that they can be mixed and then tested together for binding to an antibody sample and for viability. The two cell lines differ only by the surface antigen to which the antibodies are directed, so the differential labeling cannot use a surface antigen. My cytometer is a FACSCalibur, with standard optics (488 nm excitation, and detection filters FL1=530/30, FL2=585/42, and FL3=650LP). A recent paper (Yang, E-P, et. al. 2002. BioTechniques 32:678-682) used the dyes CMFDA and CMTMR (Molecular Probes) for differential cell labeling in the FL1 and FL2 channels, and a surface antigen was detected with a Cy-chrome fluorophore (BD) in FL3. Live/dead discrimination was not attempted in this paper, and the difference in FL3 signal between positive and negative cell lines was not very large. Presumably a cell line labeled with a single dye could be mixed with an unlabeled line to achieve the same purpose as using the two dyes, leaving one channel available for live/dead discrimination. Does anyone have an idea for a good combination of fluorophores that would allow me to do this with minimal bleedthrough? It is particularly important that the surface antigen fluorophore is well separated from potential bleedthrough from the other two dyes, since it will most likely be the dimmest of the three signals. Would the following be reasonable: Alexa 488 in FL1 for the surface antigen, CMTMR in FL2, and 7-AAD in FL3? What about CMFDA or BCECF in FL1, PE in FL2, and 7-AAD in FL3? Are there any good generic cell dyes for FL3? Any suggestions would be welcome. Thanks, Laird Bloom Phylos, Inc. 128 Spring St. Lexington, MA 02421 tel. (781) 862-6400 ext. 253 fax (781) 402-8813 www.phylos.com
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