In article <gustilo-2303951334480001 at renal3.med.upenn.edu>,
gustilo at pobox.upenn.edu says:
>>hi,
>>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.eduI 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.
