Cell kinetic effects of incorporated 3H-thymidine on proliferating human lymphocytes: flow cytometric analysis using the DNA/nuclear protein method.

Phytohemagglutinin-stimulated human peripheral blood lymphocytes incorporating high concentrations of 3H-thymidine accumulate in G2 and show a consequent reduction in the number of cells entering M (division delay). The simultaneous flow cytometric analysis of DNA content (propidium iodide fluorescence) and nuclear protein content (fluorescein isothiocyanate fluorescence) allows for the accurate quantitation of these events; G2 and M are separated in the bivariate distributions. A good correlation was observed between mitotic indices, quantitated by manually counting mitotic cells, and integration of the M area in DNA/nuclear protein histograms.

Flow cytometric analysis of RNA content in different cell populations using pyronin Y and methyl green.

Pyronin Y (PY) was used, in flow cytometric (FCM) systems, to estimate the RNA content per cell in formalin fixed EL4 leukosis tumor cells, enzyme dispersed R3327-G rat prostatic adenocarcinoma cells, mouse spleen cells stimulated with concanavalin A, and human peripheral blood lymphocytes stimulated with phytohemagglutinin. Preincubation of the cells with methyl green (MG) blocked PY binding to DNA such that the intracellular fluorescence from MG-PY was due primarily to its binding to RNA. Treatment of the cells with ribonuclease resulted in a 3- to 5-fold reduction in the fluorescence intensity of intracellular MG-PY.

DNA polymerase activity in muscle cultures.

Nuclei within myotubes do not synthesize DNA for replication. Accordingly, cultures of myotubes display low levels of DNA polymerase activity. The coincidental decline in DNA polymerase activity and increased formation of multinucleated myotubes during culture does not prove that the loss of capacity to synthesize DNA is a consequence of fusion.