help! 293F cell line

David Ko dko at bccrc.ca
Fri Jan 18 17:44:12 EST 2008


Hi Nancy,


I handled 293F and 293T (suspension) cell lines for over 2.5 years and I hope I can help.
 I haven't had any problem with using 293T or 293F to express soluble proteins, but I
like to use 293T better because the 293F does not adhere well.	The media I use for
growing 293T/F is the Freestyle media from Gibco, the % FBS is 2%, and the % CO2 I use in
the incubator is 5%.  As for the cell debris, I do see some from time to time (but not a
lot and not too frequently).  Try changing media when that happens again, because that
definitely helped me.  By that I mean changing your culture media to fresh media, and NOT
to throw away your stock bottle and replace with a new bottle/batch.

How often do you passage or split your cells?  I usually keep them at around 70%
confluent for 293F and around 500,000cells/ml for 293T (with rocker/shaker).  Also, I
split my 293T three times a week.  Yes, it is a lot of work but I have never ever had
dramatic or unexpected cell deaths.  In fact, they just wouldn't stop growing!	

Anyway, I hope that helps and I'll definitely email you if I can think of anything else.  
Thanks,

David 

Research Assistant II

Flow Cytometry Core Facility

BC Cancer Research Centre, Vancouver, Canada 



-----Original Message-----
From: Nancy Hurtado-Ziola [mailto:nhurtado at UCSD.Edu] 
Sent: Thursday, January 17, 2008 5:42 PM
To: cyto-inbox
Subject: help! 293F cell line


Hello fellow flow-ers, 


I have a soluble protein that's really difficult to express.  I mentioned it to someone
and they said I should use 293F cells to solve this problem.  Well, I need some
information on these cells.  I'm having trouble growing 293F cells.  I purchased them
directly from Invitrogen.  The first batch started out OK.  I did everything the supplier
said to do (use their SF media, use warmed media, keep cells at 37deg c, with 8% CO2
level, check viability with tripan blue, etc., etc.)  but the cell population diminished
and there was so much debris.  I got to pass 3, but never had enough cells after that to
do another pass let alone freeze down or for a transfection.  I've never seen a photo of
what these cells are supposed to look like after the culture takes off.  Do cells in
suspension typically have a lot of debris? 


I called Invitrogen and asked for help 3 different times during my attempt to grow these
cells.	They finally sent me a new vial of cells.  Again, the cells started out ok.  The
first pass, went ok, but now the cells seem to be dying and there is an increase in
cellular debris. (I also thought it might be the media so I gave them the lot number, but
there didn't seem to be any reported problems.)  Has ANYONE seen this before?  If so, did
you figure out what the problem was?  Were you able to resolve it and get a good culture
going?


As always, I appreciate any help you might have to offer.

________________________

Nancy Hurtado-Ziola, Ph.D.

Varki Laboratory


Department of Cellular and Molecular Medicine

University of California, San Diego

9500 Gilman Dr.

CMM-E 1087, Mail Code 0687

La Jolla, CA 92093

Lab Phone: (858) 534-1346

Fax: (858) 534-5611







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