Hello flow people, I'd like to purify single cells from my mouse lymphocyte cutures for PCR and RT-PCR analyses. Unfortunately, our B-D FacsCalibur is not configured for single cell sorting. It puts the sorted cells, and a constant flow of sheath fluid, into 50-ml conical tubes. The B-D engineer I just spoke to tells me they don't make any single-cell sorting module for FACSCalibur, and buying a different sorter is just not an option at the moment. So here are some wild ideas: 1. Run the cells really slowly; learn the time/sheath volume between the appearance of the cell on the monitor and its fall into the test tube; catch the cell in a smaller vessel. 2. Just sort the cells the usual way, then do limiting dilutions to hopefully obtain single cells. 3. Ask for help from owners of more sophisticated cell sorters in St. Louis ( I know you're reading this!). Any suggestions, ideas, hints? Has anyone out there gotten reliable single cells from sorting on a FacsCalibur? Thanks for any and all ideas! Lynn Lynn B. Dustin, Ph.D. Assistant Professor Molecular Microbiology and Immunology St. Louis University School of Medicine 1402 S. Grand Blvd. St. Louis, MO 63104 phone: 314-577-8415 or -8420 fax: 314-773-3403
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