Resuscitating a Hybridoma line

pogo crm at iquest.net
Sun Mar 26 01:36:21 EST 1995

In article <gustilo-2303951334480001 at renal3.med.upenn.edu>, 
gustilo at pobox.upenn.edu says:
>I'm trying to resuscitate a hybridoma line that we have. I usually grow
>the lines in DMEM HG, 10% FCS, HT, Non-Essential AA, Sodium Pyruvate,
>HEPES. The other lines grow well in this medium but there is one in
>particular that just won't grow.
>The line grew fine in the usual medium and was then frozen in LN2 (3-4
>years ago; before my time). Cell viability (by Trypan Blue exclusion) was
>fine on thawing. Now, it just won't grow in our usual medium.
>Are there any other supplements I should throw in to kick start this
>particular line?
>I have gotten suggestions to add CEGABA, T/B cell growth factors (IL2, 4,
>& 6), conditioned media.... etc.
>Which supplement worked the best for you? I'm running out of vials of this
>line and I can only try a supplement or two.
>thanks very much in advance.
>Standard Disclaimer:
>    I am not responsible for comments that I make after 3:00 A.M. or after 
5 cups of double espresso, whichever comes first.
>gustilo at pobox.upenn.edu
I would try as many things as possible all at once if you are 
desperate,,,add IL-2, Il-12, GMCSF and up the FBS to 15 or 20%
You might want to try using IMDM and since some lines prfer a more acidic 
medium try a small flask at 10% CO2.  Also some cells simply take a long 
time to recover from freezing--if only a few cells are viable you may need 
to put the flask in the back of the incubator for about 2 weeks before you 
see them acively growing.  It sounds silly, but it has saved me several 
times.  To really help you, I would have to know the parental line--that is 
your best clue to what is needed.  Good luck from IU research.

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