Abstract
Standardization and improvements upon a previously reported method (Fukuda et al. 1975) for the cytofluorometric quantitation of the hemoglobin (Hb) and nuclear DNA contents of a single erythroid cell were described. Intracellular Hb was converted to fluorescent porphyrin by irradiation with violet light (λ 405nm, fluence rate, 12×10-2mJ/sec/mm2) in the presence of SH-donor (mercaptoethylamine hydrochloride, MEA). The intensity of porphyrin fluorescence increased with prolonged photochemical reaction reaching a plateau level at 40min (total 288mJ/mm2). With this improved method, maximum fluorescence for erythroid cells was found to be about 15 times stronger in intensity than with the previously reported method.
Under maximum conditions a linear relationship was found between fluorescence intensity and Hb content (MCH) through the use of the peripheral erythrocytes of different adult male animals (sheep, man, and chick).
The fluorescences of porphyrin and Feulgen DNA were measured without interference with each other, through analysis of the emission spectra on a erythroblast lightly stained with pararosaniline Feulgen after the optimum photochemical reaction.
