SIMULTANEOUS MEASUREMENT OF NUCLEATED CELL COUNTS AND CELLULAR DIFFERENTIALS IN RAT BONE MARROW EXAMINATION USING FLOW CYTOMETER

Abstract

The purpose of this study was to establish the simultaneous measurement of nucleated cell counts and cellular differentials in rat bone marrow examination. The bone marrow cells were stained with an anthraquinone fluorescent DNA stain (DRAQ5) and fluorescence-labeled antibodies, and were analyzed quantitatively using a flow cytometer in the presence of internal standard beads. DRAQ5 distinguished populations of nucleated cells. The absolute counts of nucleated cells were determined using an internal standard, and were equivalent to that measured by the electrical resistance method. The population of nucleated cells was classified into myeloids and erythroids by labeling with CD11b/c and CD71 antibodies, respectively. In a separate examination, T- and B-lymphocytes were also classified by labeling with CD3 and CD45RA antibodies, respectively. The classification of each cell lineage was identical with that of the alternative flow-cytometric method in which cells were differentiated according to cellular size and the fluorescence of a peroxidase indicator, 2',7'-dichlorofluorescin. The ratios of cell lineage, together with myeloid/erythroid ratio (ME), were the same as those obtained by a manual microscopic method. The present flow cytometric method enables the simultaneous measurement of the total nucleated cell counts and cellular differentials of rat bone marrow cells, allowing for rapid and highly quantitative bone marrow examination in rats.