In article <359A7617.712F at mail.tju.edu>,
mark <mark.haynes at mail.tju.edu> wrote:
>John Ladasky wrote:
>>>> 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...
>>>> Well, it's official -- the 48-well culture went quiescent at the
>> beginning of last week and did not restimulate this week.
>>I hope your lines are strugglin' along. The reason for the suggestion about
>the il2 is in reference to activation in duced apoptosis that has been
>elucidated by ab. abbas for one. Have you tried the rest steps (?) maybe
>IL-4 instead of IL2, I've never wanted long-term cells so my suggestions are
>really guesses.
>markH
As of this week I have a few cultures still growing from my first
attempt, and I've started a second pass with two donors. What's working for
me at the moment? It seems that splitting to multiple wells on a 96-well
round-bottom plate, before moving to something bigger, might work. I've
tried withholding target cells this week from some cells -- that also seems
to help. I have kept in the 20 U/ml IL-2. Actually, I should be more
specific: I'm using medium supplemented with 10% Lymphocult, which is a
medium conditioned by a human T cell line. Lymphocult is standardized at
200 U/ml IL-2, but probably contains other cytokines as well.
Any reference to progress or improvement that I make is anecdotal,
of course. I could spend six months trying to optimize the CTL line pro-
cedure -- and my boss would be most displeased. On the other hand, if I
can squeeze out a few lines with my present knowledge and approach, I can
try a few experiments. At the end of this week I plan to Ficoll the two
cultures that I've expanded to four wells on the 96-well plate, and move
them to a single well on a 48-well plate. These will then receive target
cells and autologous PBMC. Seven days after that I plan to try some kill-
ing assays.
Are the cell lines that I want to grow "long-term?" Well, let's
see. I need enough cells to do the following:
a) perform killing assays against six targets at a few E:T ratios
(say 1:1, 5:1, 25:1)
b) check for CTL cell surface phenotype by flow cytometry (specif-
ically, I want to know whether cells are CD3+/CD8+/CD4-/CD56-)
c) if the cells have an interesting phenotype, have enough to freeze
some down, which I can hopefully use later to identify the exact
MHC molecule and antigenic peptide recognized by the CTL
I'm guessing that I can accomplish a) and b) with a few million CTL.
I should be there soon with a few lines. Objective c) is the tough one. I
understand that recovering CTL lines from a freeze is dicey.
--
Rainforest laid low.
"Wake up and smell the ozone,"
Says man with chainsaw. - John Ladasky
