HELP! CTL lines won't expand!
mark.haynes at mail.tju.edu
Tue Jun 23 12:48:12 EST 1998
John Ladasky wrote:
> Hi folks,
> Yesterday I looked at *nine* protocols for CTL line expansion.
> It seems like some of the primary culture steps in our protocol (days
> 0-14) are a bit unusual (and I think I understand the reasons for these),
> but nothing in the secondary steps seems to be that unusual. And the
> secondary cultures are where I'm getting my problems anyway. _Current_
> _Protocols_in_Molecular_Biology_ suggests that one should alternate be-
> tween providing stimulators with the weekly PBMC and withholding the stim-
> ulators. They also suggest regular Ficoll purification of the live cells
> away from the dead cells. None of the papers I read went to such extreme
> measures -- and besides, is it practical to do 30+ Ficolls at once?
> Am I over-stimulating my cells, leading to apoptosis? Am I under-
> stimulating them? What's up?
John, as a first guesss i would say possibly--have you tried to reduce or
eliminate the il-2 when you transfer the cells to the larger wells?-if they
are proliferating as per microscopic exam then the Il-2 may be excessive. as
you probably know blasts are quite fragile. the ficoll question is pertinent
but i wouldn't think it pertains until you are harvesting form the 48 well
plates but that is another easy question to address--good luck
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