Dear Flow Folks, We are attempting to use CFSE to monitor in vitro MLR?s for a study of alloregulatory cells in mice. Our model involves a relatively weak response (single class plus minor antigens). Our responder cells are labelled with CFSE and, when harvested, stained with either CD4- or CD8-Tricolor (Fl3). We take great care with compensation, as the bright CFSE fluorescence spills into Fl2 and Fl3. We live gate on lymphocytes/blasts using FSC/SSC. Our problem is with autofluorescence of the cells that are not labelled with CFSE, stimulator cells, and particularly, the cells we are adding as co-cultured cells: responder strain regulatory cells or responder strain naïve cells (controls). The co-cultured cells are added as unfractionated or fractionated splenocytes. The autofluorescence of the non-CFSE labelled cells increases during 72 hr in culture until it spans 2 decades on the Fl1 log scale. The brighter autofluorescence thus interferes with measuring the number of responder cells that have undergone 4 or 5 to 8 divisions and are then CFSE dim. Thus the usefulness of the CFSE measurement is severely limited. Our responder cells are washed well before culture and reutilization of the CFSE label does not appear to be a problem. In contrast to the in vitro MLR, in vivo MLRs performed with CFSE give us beautiful data (but unfortunately are not suitable for our regulatory cell study). We would be grateful for any information or thoughts on this problem you may have. Many thanks, Sally De Fazio Albany College of Pharmacy & Northeastern University
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