I've been working for 15 months on a simple CTL assay that doesn't seem
to work. Here's a brief run-down on what I've been doing...
System: Chromium release
Animal: Murine lymphocytes from spleen- either Balb/c, B6D2F1, or
C57BL/6
Target: p815 (Balb/c)/EL4 (B6) infected with Type A influenza (PR8)
-infected log phase cells with 2000HA/ml for 1.25hr; after several
washes, cells were left to incubate for 6 hrs followed by 51Cr labelling
-51Cr labelling established
-CTL assay performed for 6hr with E:T ratios 200-100-50-25-12.5-6.25 in
round-bottom 96-well cell culture plates
Immune response evaluated: Primary CTL response after IP injection of
virus (300HA); no in vitro secondary stimulation
Parameters tinkered around with (parameters tested are in parentheses):
-different p815 cell lines and PR8 viruses (targets and viruses from our
lab and another lab in Florida)
-amount of virus used to infect targets (200-2000HA)
-amount of chromium used/infection protocol (100-200uCi per 1e6 cells)
-pH of infection medium (6.5-7.5)
-composition of infection medium (with or without serum)
-container in which infection of targets were performed ( conical or
cell culture flask)
-container in which target cells were grown (flask or petri dish,
treated or not treated for cell culture)
-type of media used to grow target (RPMI-1640, Iscove's DMEM or DMEM,
with or without 2-ME)
-route of primary infection of mice (IN or IP)
-after secondary infection of mice (3,5,7 days)
-after secondary culture of lymphocytes (3,5,7 days)
-duration of infecting targets (15minutes-12 hours)
-duration of CTL assay (4-12 hours)
-timing of assay post-infection of mice (3,5,7,10,12 days)
As you can tell, I've tried several parameters and I'm absolutely
frustrated....I think I've narrowed it down to the idea that my target
cells are NOT being infected with flu virus in vitro, despite the same
virus being infectious in vivo (IN administration of an appropriate dose
kills mice)
Is there anyone or any lab that has a CTL assay working and is willing
to have someone visit and figure out what's going wrong in our lab? At
this point, I've incorporated or tested as many suggestions as possible
without any luck- right now, I'm willing to spend my own time and money
to visit someone anywhere in the US who has the same assay currently
functioning.
--
*******There's got to be a better way!!!*******
John Leander Po, M.Sc.
Professional student
MCP Hahnemann School of Medicine
po at drexel.edu
215-991-8369
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