Hi Lynn, I've had a similar experience but a different kind of assay. We were doing phagocytosis assays with Mosquito cells and fluorescent bacteria. The results on the microscope were very different to what we were getting on the Facscan- Anyway what was happening was that these cells had loads of vacuoles which were also fluorescing in the green channel. You could see this on the microscope- and in the end we gave up on the FC. You might have a similar problem - so by microscopy you need to see if other components of the cells are also fluorescing e.g yellowish, orangy, greenish etc. I would also suggest decreasing the p-formaldehyde concentration if possible as it may also contribute to "unwanted fluorescence". Switching to Fitc as a marker might be an option as from personal experience I find Alexa 488 to be very bright (for HeLa- PMT 427) and as you say the cytosolic antigen expression is a punctate distribution- maybe the PE signal is simply too weak when used in conjunction with a very bright marker?? Could that happen, flowers?? Have you compared -by microscopy- the sample prepared for FC and the sample prepared for microscopy? can't think of anything else regards Ann
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