Differences in chromatin thermal lability between thymic and splenic lymphocytes in intact and adrenalectomized rats detected by acridine orange microfluorometry

The sensitivity of chromatin to thermal denaturation was compared between small lymphocytes from the thymus and spleen of intact and adrenalectomized rats. RNA was enzymatically removed from uniformly spread lymphocytes attached to glass slides in low density and chromatin denaturation effected by heating at 90 °C in a solution free of formaldehyde and sodium ions. The preparations were stained with acridine orange following acetylation. Fluorescence emission intensity of the nuclei in individual cells was measured at 530 and 590 nm and the ratio used as a relative index of chromatin denaturation. The data show that the chromatin of small thymus lymphocytes is generally more thermolabile than that of morphologically comparable cells of the spleen in both intact and adrenalectomized animals. This difference between cells of the same morphological type from the two lymphoid organs was not evident from measurements of the amount of dye bound by the cells without denaturation. Removal of the endogenous source of glucocorticoids resulted in an increase in the number of spleen lymphocytes with lower chromatin thermal stability but had no detectable effect on the population of thymus lymphocytes. The results are discussed in terms of the histological organization and immunological role of the thymus and spleen and in relation to the technical aspects of acridine orange microfluorometry as a sensitive cytochemical probe of chromatin structure.