Hello Kenichi, I've done quite a lot of cell cycle sorting over the years, and have had similar problems with small numbers recovered from cell cycle sub-components. The best strategy that I have developed is as follows: i) Always use polypropylene collection tubes -anything else, and the cells will stick irretrievably. ii) Match the collection tube size to the number of cells recovered i.e. use the smallest possible tube - 1exp5 tubes fit into a 2ml screw-top 'ependorf' tube, 1esp6 fit into a 15ml tube. iii) Pre-block the tubes with some 10% FCS prior to use. iv) Collect the cells directly onto a cushion of neat FCS -I have never had any problems with cell viability, and I must have sorted dozens of cell lines and primary cells this way. v) After collection, centrifuge samples as following: 2ml tubes-400g 5mins, 15ml tubes-400g 15mins. vi) For very rare events, use a grounding wire in the collection tube. vii) Try to arrange for the sort stream to land in the FCS -not the side of the tube. viii) I have also tried siliconizing the collection tubes -though I am unsure whether this makes a difference. Best regards, Arnold 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, _/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/ Arnold Richard Pizzey Department of Haematology Royal Free and University College London Medical School 98 Chenies Mews London WC1E 6HX U.K voice: +44 020-7679-6234 Fax: +44 020-7679-6222 email: a.pizzey@ucl.ac.uk _/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/_/
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