27
Ficoll isolated cells from anterior kidney cell suspensions and in the
stimulation indices with mitogens. Furthermore these differences ap
peared to be correlated with the length of time the bluegills were main
tained in holding tanks in the laboratory prior to sacrifice. Table 3
presents data on white cell differentials of Hypaque-Ficoll isolated
cells from anterior kidneys of fish sacrificed at either one day or
three weeks after capture. Significant increases in the number of blast
like cells were seen in cell preparations from bluegills maintained for
three weeks. The gross differences in the cell types isolated from
Hypaque-Ficoll can be seen by comparing the cells shown in Figure 2
(from a one day isolate) with those in Figure 3 (a three-week isolate).
An increase in the number of red cells, which would not penetrate the
Hypaque-Ficoll, was also noted in the three-week isolates.
Furthermore, it was also observed that in experiments utilizing
fish maintained in acquaria for long periods of time, TCA precipitable
counts of control unstimulated cultures were high. A composite of data
from five experiments in which bluegill were sacrificed at various
periods of time 'after capture is presented in Table 4. Apparently the
longer a fish is maintained under our laboratory conditions the more
likely it is the animal's lymphocytes will exhibit a high level of
spontaneous thymidine incorporation. It thus seems imperative to use
freshly caught fish as sources of cells for in vitro studies if the
alternative is to keep them under the conditions used here.
To determine if TCA precipitable counts were a valid measure of
cellular events in culture, TCA precipitable counts were correlated
with the actual number of cells containing labeled thymidine. The
technique of autoradiography was used. Antei'ior kidney lymphocytes