CYTOFLUOROMETRIC MEASUREMENT OF CONTENTS OF NUCLEAR DNA AND INTRACELLULAR PORPHYRIN CONVERTED FROM HEME OR HEMOGLOBIN ON A SINGLE ERYTHROID CELL

Abstract

A technique for cytofluorometric measurement of contents of Hb and nuclear DNA on a single erythroid cell is described. Smears of mouse bone marrow were fixed with methanol and treated with 0.2 M mercaptoethylamine hydrochloride (MEM) dissolved in 1.5 M perchloric acid, irradiated by UV light under a fluorescence microscope. This is a modification of method of Granick et al. (1965) to convert hemoglobin into fluorescent porphyrin. The smears were then stained with Feulgen nuclear reactions. The non-specific fluorescence in the background was eliminated by pre-, or post-irradiation method with the wavelength 543 nm (Fujita and Fukuda, 1974). The amounts of Feulgen nuclear DNA and intracellular porphyrin converted from heme or Hb were determined by cytofluorometric measurement on a single erythroid cell. With the two quantitative parameters. erythroid cells in bone marrow were classified into seven classes of different maturation stages. “Proerythroblasts” which were identified on the bases of morphological criteria had in general aneuploid amounts of nuclear DNA with disproportional contents of Hb showing rather aberrations from normal steps of cell maturation. The DNA amounts of “orthochromatic erythroblasts” showed continuous decrease from diploid range to almost zero suggesting that the removal of nuclear DNA is not exclusively due to mechanical expulsion of a whole intact nucleus.