Happy New Year, I too would rather sort CHO cells than HEK 293. However, I have had good luck ~20-30% by gently centrifuging the 96-well plates to "stick" the cells to the plastic. I use ~100-200g for 2-3min. My results with CHO cells is correspondingly satisfactory. Regards, Dave G. David F. Gebhard Jr. Senior Scientist EMS-New Leads FACS Facility Pfizer, Inc. Eastern Point Road MS 8118W/ 144 Groton, CT 06340 TEL 860-715-0830 FAX 860-441-6410 -----Original Message----- From: Hastings, Richard C [mailto:richard.hastings@astrazeneca.com] Sent: Wednesday, December 18, 2002 10:57 AM To: cyto-inbox Subject: Sorting HEK 293 and establishing clonal cell lines Hi, This message is related to the CHO cell sorting thread. I perform many sorts with HEK 293 and CHO transfected with different receptors. I generally perform single cell deposition into each well of 96 well plates to establish clonal cell lines. My problem is getting these single cell clones to grow especially with the HEK 293 (I have much better success generating CHO lines). I have tried all sorts of little tricks to get these cells to grow such as using conditioned media in the wells, and using collagen- or poly-D-lysine-coated plates. My success rate with single cell deposition with HEK 293 is quite poor, I am estimating its probably below 1%. Meaning, I am lucky to generate one clone per 96 well plate. Of course, success depends quite a bit on the gene transfected into the HEK 293 and the health of the cells at the time of sorting. Does anybody know of any conditions that would increase the number of clones I could generate using HEK 293? Thanks, Rich Richard C. Hastings Associate Scientist Target Expression/LDD 1800 Concord Pike Wilmington, DE 19803 302-886-2452 richard.hastings@astrazeneca.com LEGAL NOTICE Unless expressly stated otherwise, this message is confidential and may be privileged. It is intended for the addressee(s) only. Access to this E-mail by anyone else is unauthorized. If you are not an addressee, any disclosure or copying of the contents of this E-mail or any action taken (or not taken) in reliance on it is unauthorized and may be unlawful. If you are not an addressee, please inform the sender immediately. -- End --
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