24
conditions in a rather "piecemeal" fashion over an extended period of
time. The following commentary is an effort to systhesize the major
observations that enabled the definition of what can be called optimal
conditions. Unless otherwise noted all of these studies were performed
with anterior kidney lymphocytes.
Various sera or plasma were tested to determine which was a suit
able supplement to use with RPMI 1640 for mitogenic studies of cultured
bluegill cells. Ten percent human, calf, fetal calf, rabbit, alligator,
bass, catfish, and grouper sera or plasma and mixtures of 5% humanserum
with 5% calf or fetal calf serum were not supportive in mitogenic stimu
lation studies using PHA (0.1 yl), Con A (10 yg) or LPS (1 or 10 yg) at
either 22, 27 or 32C. Grouper and catfish sera were cytotoxic for
bluegill cells. The other sera gave high TCA precipitable counts in
unstimulated control cultures and stimulation indices for mitogen
stimulated cultures of 1 or < 1. On the other hand, bream (a collective
term for all Lepomis species) serum pools were supportive in the sense
that significant stimulation indicies were obtained with mitogen stimu
lated cultures. '
In the initial experiments 10% bream serum was used. However, due
to the limited supply of bream sera and the difficulty in obtaining
good yields of serum from clotted blood, two modifications were tried
and found satisfactory; 1) heparinized plasma rather than serum was used
and 2) the concentration of supplement was reduced from 10% to 7%.
An additional complication was the observation that not all bream
plasma pools were suitable as supplements in mitogenic assays. Varia
tions in TCA precipitalbe counts of unstimulated control cultures ranged
from < 100 CPM to > 10,000 CPM and stimulation indices varied from 4 to