Evaluation of Mn2+ stimulated and Zn2+ inhibited apoptosis in rat corpus luteal cells by flow cytometry and fluorochromes staining.

We had reported that Mn2+ is the most effective divalent ion in stimulating apoptosis and Zn2+ inhibits apoptosis in corpus luteal (CL) cells of rat, which evidenced by the appearance of internucleosoma DNA fragmentation in electrophoretic gel. To further understand morphological character of apoptosis undergone in CL cells, we performed fluorochromes staining and flow cytometric studies on Mn2+ and Zn2+ treated CL cells. Cells dispersed by collegenase-DNase from PMSG-hCG primed pseudopregnant rats were incubated in KRBGAL solution for 4 h with 5 mM Mn2+ or Zn2+. Staining of nuclei with the DNA binding fluorescent dyes, acridine orange-ethidium bromide (AO-EB), showed an increased green fluorescence of AO in Mn2+ treated cells as compared with control or Zn2+ group. DNA content analysis showed that Mn2+ caused an absolute shift from right to left side in the scatter profile by Flow Cytometry. The consistency of above results supported that Mn2+ is the most effective divalent ions in stimulating apoptosis in CL cells of rat, and provided a morphological character of apoptosis undergo in CL cells. The results also suggest that fluorochromes staining and flow cytometry are effective and convenient methods in study of apoptosis in CL cells.