HELP! CTL lines won't expand!

John Ladasky jladasky at pmgm.Stanford.EDU
Thu Jun 25 16:47:00 EST 1998


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



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