help! 293F cell line

Gilman-Sachs, Alice Alice.Gilman-Sachs at rosalindfranklin.edu
Wed Jan 23 15:47:20 EST 2008


Mycoplasma

 

 

________________________________

From: Joel Tabb [mailto:jtabb at agavebio.com] 
Sent: Monday, January 21, 2008 11:44 AM
To: cyto-inbox
Subject: RE: help! 293F cell line

 

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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