I want to separate breast tumor from mouse model of breast cancer and maintain it to do flow cytometry tomorrow of that day to detect macrophages ,what is your idea about maintenance conditions that i do it?
maybe one or two day!
Cut the tumor into small pieces (about 1 mm3) then use an enzymatic digestion cocktain (collagenase I and DNase I may be sufficient). Filter the suspension to remove cell clumps or aggregates from single cell suspension. Wash several times to remove debris. It is better to label freshly obtained macrophages. But if you have to maintain them for some time then put them in culture (full RPMI). Do not forget that these cells are strongly adherent, so you will need to scrape them off the tissue flask to analyse on flow cytometry. Surely you will loose many cells by maintaining and scraping...
Hope this will help.
The cells are very sensitive and for the best results you should do the prep same day as your cell sorting. If you have to make a break (for what ever reason) I would suggest to do it at the last stage, once you have finished your FACS prep and you have all your samples ready to go in FACS solution, wrap your tubes in aluminum foil (protect from light) and store it in the fridge at +4C for overnight. In this case you can do data acquisition the next day. Best of Luck!
I agree with Gunes that you will probably loose many cells by maintaining in cell culture, but it may work.. Another option that I could think it is to isolate the cells, resuspend them in FBS + 10%DMSO, and freeze them down using a controlled rate freezing apparatus in -80oC, until FACS. Good luck!
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