help! 293F cell line

Joel Tabb jtabb at Agavebio.com
Mon Jan 21 12:44:08 EST 2008


Nancy,

  We are having almost identical problems with 293F cells from Invitrogen.
Like you, we have contacted Tech support several times, and gotten different
answers each time.  Once they said that multiple groups have complained and
they traced it down to a problem with a specific lot of media.	Alas, they
have replaced our cells 3x and our media 2x, and still no improvement.	We
have given up and gone back to adherent 293 cells.  We have started to
obtain cells that express more protein the old-fashioned way-carefully
trying different expression vectors and patiently screening/selecting high
expressing clones.  It's been slow and tedious, but we are finally starting
to get results.


Regards,


Joel Tabb, Ph.D.

Principal Scientist

Agave BioSystems

401 E. State Street

Ithaca, NY  14850

607-272-0002

www.agavebio.com


  _____  

From: Nancy Hurtado-Ziola [mailto:nhurtado at UCSD.Edu] 
Sent: Thursday, January 17, 2008 8:42 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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