In article <358FEA5C.25B5 at mail.tju.edu>,
mark <mark.haynes at mail.tju.edu> wrote:
>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.
Hi, Mark,
O.K., so you're suggesting that when I see healthy-looking, active-
ly-clumping cultures at the end of a week, I should provide PBMC and target
cells, but withhold the IL-2? I could give this a try, but I'm a bit sur-
prised. Some of the protocols I've seen call for 50-100 U/ml IL-2, and I'm
only using 20 U/ml. Of course, one other protocol calls for 10 U/ml IL-2
and 50 U/ml IL-4...
>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
Well, it's official -- the 48-well culture went quiescent at the
beginning of last week and did not restimulate this week.
>markH
--
Rainforest laid low.
"Wake up and smell the ozone,"
Says man with chainsaw. - John Ladasky
