21
blood, spleen, anterior kidney, and thymus of bluegill. Less than 5% of
the total number of cells recovered from Hypaque-Ficoll were RBC's and
the number of lymphocytes recovered represented at least 99% of the
lymphocytes present in unfractionated whole blood or lymphoid organ cell
suspensions.
White cell differentials of whole blood before and after fractiona
tion on Hypaque-Ficoll are presented in Table 1 and illustrate the effi
ciency of this technique in removing other cell types. Figure 2 is a
photomicrograph of the type of lymphocyte preparations routinely obtained
from blood or lymphoid organ cell syspensions. These cell separations
were successful, only if freshly caught fish were used. Another major
cell type, a lymphoblast-like cell, was isolated from Hypaque-Ficoll if
cell suspensions from fish maintained in laboratory aquaria for long
periods of time were used (see Figure 3). The relevance of these blast
like cell isolates and the necessity of using newly acquired fish for
these and subsequent studies is discussed in a later section.
The anterior kidney was the most abundant source of lymphocytes
' 8
(yielding 'u- 2 x 10 cells/fish) whereas spleens and thymuses routinely
7 7
yielded about 5 x 10 and 2 x 10 cells/fish, respectively. Heparinized
blood yielded about 5 x 10^ cells/ml (see Table 10).
Culture Conditions and Assay of Cell Division
As in any study involving in vitro culturing of lymphocytes (or
any other cell type for that matter) there were numerous variables to
be considered. In light of the fact that relatively limited numbers of
cells were available from individual fish and since syngeneic bluegills
were not obtainable it was necessary to approach optimization of culture