Hi! I wish to perform FACS of cells isolated from a subcutaneous tumor. Kindly suggest methods that would be suitable for making single-cell suspension of the tumor excised from mice
I would suggest to use collagenase (with or without DNase) in RPMI with 2% FCS to digest chopped tumors at room temperature for 20 to 30 min, the mixture should be mixed up and down using Pasteur pipette continuously. This should be followed by passing the digested stuff through cell strainer and washing. Add EDTA (1 mM final concentration) to your digested mixture to prevent cells clumping. I hope this helps.
Hi Shraddha, there are many ways to dissociate cells using proteinases, but the downside is that you will probably lose most surface antigens.
If you have internal markers then you are probably OK.