Hi everyone: I have been evaluating a cell cycle specific expression of various proteins. In stead of synchronizing cells using double thymidine block, etc, we are attempting to sort cells and perform Western on the sorted cells to evaluate the expression levels of proteins of interest. We are using U2OS cells. We propagate U2OS cells so that next day, cells will be 70-80% confluent. We harvest them by trypsinization and fix them overnight with 70% MetOH. Next day, we stain DNA with propidium iodine and take the cells to a core facility. Our problem has been that we only get 0.5 million G0/G1 cells out of 30 million cells we start with. The counter on the machine says 1.0 million but when we count them by hemocytometer, the number is always lower. Because of this low yield, our Western blot has not been able to detect consistent signals of the proteins. Has anybody tried a similar approach? How would you increase the yield of sorting? What do you think is causing the discrepancy between the machine read-out and actual cells sorted (as assessed by hemocytometric counting). Thank you very much for your time and help on this. Sincerely, Ken
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