Ray, My experience with 293 (human embryonic kidney, from my records), is that they are rather sensitive and can be tricky to culture in bulk, let alone low density seeding. I did find that they grow well in DMEM with 10% FBS (bulk cultures, not single cell/well). You might try seeding them into media containing 20% FBS for the first week or two, then weaning back to 10%. I have done this in the past to give a limiting dilution seed a boost. You might also try pre-coating the wells with 0.01% gelatin to help the cells adhere after FACS seeding (the matrix seems to improve their overall viability). Shari Brezinsky Biogen Cambridge, MA "ray hester" <rhester@jaguar1.usouthal.edu> on 06/28/2000 02:14:52 PM To: cyto-inbox cc: (bcc: Shari Brezinsky/Cambridge/Biogen) Subject: Flow-cloned cells Hi, For those who have experience cloning cells, what do you do to insure that single cells, cloned by FACS into 96-well plates, continue to grow? Add factors (if so, which) or cell growth-conditioned medium? Although any suggestions in general will be appreciated, the cells in question at the moment are 293 cells (human kidney carcinoma, I believe). Any advice would be appreciated. Thanks. Ray Raymond B. Hester, Ph.D. Research Cytometry Laboratory CSAB 357, Biotechnical Services University of South Alabama Mobile, AL 36688 Voice: (334) 460-6029 FAX: (334) 460-6073 E-mail: rhester@jaguar1.usouthal.edu
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