Flow cytometric determination of mitochondrial membrane potential changes during apoptosis of T lymphocytic and pancreatic beta cell lines: Comparison of tetramethylrhodamineethylester (TMRE), chloromethyl-X-rosamine (H 2-CMX-Ros) and MitoTracker Red 580 (MTR580)

Abstract

The mitochondria-specific dyes, TMRE, H 2-CMX-Ros and MTR580 were determined for their suitability to measure mitochondrial potential changes of the T cell leukemia cell line Jurkat and insulin-secreting beta cell line NIT-1 during apoptosis. Both freshly harvested Jurkat and NIT-1 cells induced to undergo apoptosis displayed poor retention of the potential-sensitive, intrinsically fluorescent dye, TMRE. Treatment with formaldehyde or paraformaldehyde completely abolished TMRE uptake in both cell types regardless of apoptosis induction. Interestingly, freshly harvested apoptotic Jurkat cells exhibited lower retention of H 2-CMX-Ros, indicating marked reduction in the oxidative status of lymphoid cells during apoptosis. This is in contrast to NIT-1 cells which failed to display significant reduction in H 2-CMX-Ros retention after anoikis induction. Paraformaldehyde treatment reduced the retention of H 2-CMX-Ros in live Jurkat cells but still allowed the discrimination of apoptotic cells which poorly retained H 2-CMX-Ros. However, live Jurkat cells lost their ability to retain H 2-CMX-Ros after formaldehyde treatment. In contrast, treatment with paraformaldehyde or formaldehyde did not have significant impact on the retention of H 2-CMX-Ros in both live and apoptotic NIT-1 cells. The uptake of MTR580 was independent of mitochondrial membrane potential in both T and beta cell lines. However, MTR580 was comparable to H 2-CMX-Ros for confocal microscopic analysis of apoptotic Jurkat cells following fixation with formaldehyde and cell permeabilization. These data demonstrate that while TMRE and H 2-CMX-Ros are suitable for determining mitochondrial membrane potential changes during apoptosis in lymphoid cells, only TMRE is suitable for such analysis in beta cells. Both H 2-CMX-Ros and MTR580 proved to be suitable for confocal imaging of mitochondria.