Wanted: ConA - rat spleen supernatant protocol

[Robyn]

Hello,

I'm trying to culture a beta-galactosidase specific mouse CTL line
(0805B) that was sent to me frozen.  The original protocol recommends
feeding it with RPMI-10 supplemented with 5% "RCAS" -- supernatant
from rat splenocytes treated with ConA.  This is supposed to contain
IL-2 and smaller amounts of other cytokines to make CTL happy.

I have tried using hrIL-2 with no luck and would like to try culturing
the cells with RCAS instead.  Unfortunately, the "Gold Book" (Current
Protocols in Immunology) tells me the concentrations of splenocytes
and ConA to use, but not how long to stimulate before harvesting the
supe!

Has anyone made this reagent before?  If anyone is willing to send me
their protocol, I'd appreciate it greatly.  My CTL and I thank you in
advance.

Robyn Reed
Duke University
Dept. of Cell Biology