Inhibition of mitochondrial respiratory chain complex I by rotenone had been found to induce cell death in a variety of cells. However, the mechanism is still elusive. Because reactive oxygen species (ROS) play an important role in apoptosis and inhibition of mitochondrial respiratory chain complex I by rotenone was thought to be able to elevate mitochondrial ROS production, we investigated the relationship between rotenone-induced apoptosis and mitochondrial reactive oxygen species. Rotenone was able to induce mitochondrial complex I substrate-supported mitochondrial ROS production both in isolated mitochondria from HL-60 cells as well as in cultured cells. Rotenone-induced apoptosis was confirmed by DNA fragmentation, cytochrome c release, and caspase 3 activity. A quantitative correlation between rotenone-induced apoptosis and rotenone-induced mitochondrial ROS production was identified. Rotenone-induced apoptosis was inhibited by treatment with antioxidants (glutathione, N-acetylcysteine, and vitamin C). The role of rotenone-induced mitochondrial ROS in apoptosis was also confirmed by the finding that HT1080 cells overexpressing magnesium superoxide dismutase were more resistant to rotenone-induced apoptosis than control cells. These results suggest that rotenone is able to induce apoptosis via enhancing the amount of mitochondrial reactive oxygen species production.

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The oxidative burst is frequently evaluated by the conversion of dihydrorhodamine 123 (DHR) to rhodamine 123 (R123) and hydroethidium (HE) to ethidium with the use of flow cytometry (FCM). Added R123 accumulates in mitochondria, but during phagocytosis R123 originating from DHR has been observed in neutrophil granules. The present study was designed to identify the site of reactive oxygen species (ROS) formation and the intracellular traffic of R123 in neutrophils by using mitochondrial membrane potential probes and the lysosomotropic probe LysoTracker Red, which have not previously been applied to neutrophils. Quiescent and phagocytosing human peripheral blood neutrophils were incubated with DHR, HE, R123, MitoTracker Green (MTG), MitoTracker Red (CMX-Ros), and LysoTracker Red alone and in all combinations of red and green probes, and studied by FCM and confocal laser scanning microscopy (CLSM). Phagosomes were filled with R123 originating from DHR. Phagocytosis also triggered the oxidative burst in oxidative response granules that differed from acidic granules. All the neutrophils stained with mitochondrial and lysosomotropic dyes.

COUNTING CELLS USING A HEMACYTOMETER Determining the number of cells in a preparation or a culture may at times be necessary for standardization of conditions, estimation of staining reagent required, or accurate quantitation experiments. Cells can be counted directly under the microscope with the aid of the hemacytometer. Cell viability can also be determined by staining cells with trypan blue and counting (see APPENDIX 3B, Support Protocol 3). The hemacytometer is a thick glass slide with a central area designed as a counting chamber. The exact design may vary; the one described here is the Improved Neubauer (Fig. A.3A.1). The central portion of the slide is the counting platform, which is surrounded by a 1-mm groove. A transverse groove divides this platform into two counting chambers, each consisting of a silver footplate on which is etched a 3 × 3-mm grid. This grid is subdivided into nine 1 × 1-mm squares. Cells are usually counted in the four corner squares and the center square. The former are further divided into 16 tertiary squares and the latter into 25 tertiary squares to aid in counting. Accompanying the hemacytometer slide is a thick, even-surfaced coverslip. Ordinary coverslips may have uneven surfaces, which can introduce errors in cell counting; therefore, it is imperative that the coverslip provided with the hemacytometer be used in determining cell number. Cell suspension is applied to a defined volume and counted so cell density can be calculated. Materials 70% (v/v) ethanol Cell suspension 0.4% (w/v) trypan blue or 0.4% (w/v) nigrosin, prepared in HBSS (APPENDIX 2A) Hemacytometer with coverslip (e.g., Improved Neubauer, VWR) Hand-held counter Prepare hemacytometer 1. Clean surface of hemacytometer slide and coverslip with 70% alcohol. Coverslip and slide should be clean, dry, and free from lint, fingerprints, and watermarks. 2. Place the coverslip over the central area of the hemacytometer and press firmly into position. The coverslip should rest evenly over the silver counting area. Some workers place a small amount of water on the ground glass area of the hemacytometer, on which the coverslip rests, to improve the seal. Prepare cell suspension 3. For cells grown in monolayer cultures, detach cells from surface of dish using trypsin (see APPENDIX 3B, Basic Protocol). 4. Dilute cells as needed to obtain a uniform suspension. Disperse any clumps. When using the hemacytometer, a maximum cell count of 20 to 50 cells per 1-mm square is recommended.

Abstract:Apoptosis permits neutrophil recognition by macrophages, thereby not only limiting potential cytotoxicity but also promoting resolution of inflammation.