We also take care not to dump the defrosted contents of the ampule into the 1:20 dilution of media...this can "explode" the cells because of a sudden change in osmolarity...thus, we add "dropwise", with gentle shaking, the 20ml of media to the contents of the ampule (which we have transferred to a 50.0 ml tube). Takes about 20 min. Works every time! --Ross-- Ross E. Longley, Ph.D. Harbor Branch Oceanographic Inst. 5600 U.S. #1, North Fort Pierce, FL 34946 -----Original Message----- From: Joost Schuitemaker [mailto:J.H.Schuitemaker@AMC.UVA.NL] Sent: Friday, January 28, 2000 3:09 AM To: cyto-inbox Subject: RE: Thawing PBMC's Dear Dagna, It is possible to get yields above 90%. The trick is to defrost the ampul until a small ice clump. And add the just defrosted DMSO/medium to 37°C warm medium in a ratio at least 1:20 (ampul:medium). First wash should be 20°C and when you are going to culture the cells keep washing above 15°C. This works perfect with human PBMC's and mouse lines. Success, Joost Schuitemaker _________________________________________ J.H.N. Schuitemaker Research Technician Dept. Cell Biology & Histology Cellular Immunology Group P.O.box 22700 1100 DE Amsterdam The Netherlands Tel. +31 (0)20 5664960 Fax. +31 (0)20 6974156 E-mail privé: J.H.N.Schuitemaker@consunet.nl -----Original Message----- From: Dagna Sheerar [mailto:dagnas@primate.wisc.edu] Sent: woensdag 26 januari 2000 20:06 To: cyto-inbox Subject: Thawing PBMC's Hello Everyone, Occasionally, I have to thaw some PBMC's for Flow. They are frozen in DMSO in 1 mL aliquots. The thawing procedure is as described on the instruction sheet that comes with the DMSO. I am wondering what kind of yeilds people get when thawing PBMC's. Is it normal to recover only 50% of the cells? These are rhesus PBMC's and are infected with HIV/SIV/SHIV. I would appreciate any information anyone could share. Thank you very much Dagna Sheerar Wisconsin Regional Primate Research Center University of Wisconsin - Madison 1220 Capitol Court Building #2 Room #215 Madison, WI 53715 608 265 3796 dagnas@primate.wisc.edu
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