13
7
White, red, and dead cells were enumerated and 1x10 viable white
cells in three ml of supplemented medium were aliquoted in Falcon 35
x 10 mm tissue culture dishes (Scientific Products).
Sheep red blood cells (SRBC's) used for immunization of the dis
sociated organ suspensions were obtained from a single sheep (Colorado
Serum Comp., Denver, Col.; Sheep # 20, H type antigen). SRBC's were
washed three times with RPMI 1640 and the final pellet suspended in the
enriched RPMI 1640 (without supplement) to 1% of the packed cell volume
Cultures to be immunized received 0.1 ml of the 1% SRBC suspension.
Controls received 0.1 ml of enriched RPMI 1640.
Culture dishes were maintained in 5% CO2 95% air humidified
environments as described above.
Assay for ^H-thymidine Incorporation into DNA
An automatic cell harvester (Otto Hiller Company, Madison, Wis.)
was used to obtain trichloroacetic acid (TCA) precipitable nucleic
acid material from individual wells of cultured cells. Twenty-four
hour pulsed cells were syphoned from the wells onto a glass fiber
fc
filter (Reeve Angel, Whatman, Inc., Clifton, N.J.), rinsed with
saline, precipitated with 10% TCA and methanol dried. Discs, repre
senting individually harvested wells, were punched out of the filter
strip and assayed for using liquid scintillation counting. The
scintillation cocktail used consisted of PPO (Packard, Chicago, Ill.;
16.5 g), POPOP (Packard; 0.3 g), Triton X-100 (Packard; 1.0 L), and
toluene (Maltinckrodt, St. Louis, Mo.; 2.0 L). Samples were counted
in mini-vials (Rochester Scientific, Rochester, N.Y.) using an auto
matic liquid scintillation counter (Beckman Instruments, Fullerton, Cal
h
) 
Model LS-100