47
The number of cells in the various lymphoid organs containing
cytoplasmic immunoglobulin were assayed by indirect immunofluorescence.
Smears of washed, unfractionated cell suspensions of anterior kidney,
thymus, spleen, blood, and posterior kidney (as a negative control)
were examined and the number of cells showing positive cytoplasmic
immunoglobulin staining quantitated. The results are presented in
Table 10. The posterior kidney was devoid of any Ig-containing cells.
Anterior kidney, spleen, thymus, and blood demonstrated appreciable
numbers of immunoglobulin containing cells. ,
Preliminary studies were undertaken to determine if bluegill lym
phoid cell suspensions would respond In vitro to an antigenic stimulus.
Several modifications of the culture techniques discussed above for
mitogen studies were employed to enrich the culture media and to ensure
that all necessary cellular components were present.
Undialyzed 7% bass plasma rather than bream plasma was used as a
supplement with an enriched RPMI 1640 medium. Since the hemolytic plaque
assay only measured differences in the number of plaque-forming cells
between control and antigen stimulated cultures, a high nonspecific stimu
lus by bass plasma (see mitogenic studies) was irrelevant as long as an in
crease in plaque-forming cells was attributable to antigenic stimulation.
A pool of unfractionated cell suspensions of anterior kidney', spleen,
and thymus was used for three reasons: 1) to increase the number of
available cells and thus the number of variables that could be tested,
2) to include phagocytic and plasma cells as well as any other cell
types possibly involved in antigen processing and antibody formation,
and 3) to decrease the chance of compartmental effects of individual