We have been trying to label CD4 T cells with CellTracker Green, from Molecular Probes, and seem to have a problem with heterogeneous loading. These cells are isolated from mouse spleens, purified by negative selection using Dynal beads and have a viability of 95-98%. When examined by FCM, there is a nice, bright green positive population but there is also a "tail", representing about 20% of the cells, which has lower and variable amounts of dye loading. Variation of incubation time and/or dye-loading concentration does not seem to overcome this phenomenon. Previous experience with dendritic cells has produced uniform loading. So, are T cells inherently different or is there some trick to get uniform loading of these cells? Any help/suggestions would be greatly appreciated. Wayne F. Green
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