Simultaneous determination of apoptosis and autophagy using flow cytometry
To explore the potential crosstalk between ALS-induced apoptosis and autophagy, we further determined the two modes of programmed cell death simultaneously. SKOV3 and OVCAR4 cells were plated in six-well cell culture plates (Corning Incorporated, Corning, NY, USA) at a density of 1×105 cells per well. After incubation overnight, the cells were incubated with fresh medium, vehicle alone (0.05% DMSO, v/v), or ALS at 0.1, 1, and 5 μM for 24 hours. For comparison purposes, the autophagy inducer SB203580 at 10 μM, autophagy inhibitor wortmannin at 10 μM, chloroquine at 30 μM, bafilomycin A at 10 μM, and the apoptosis inhibitor Z-VAD(OMe)-FMK at 20 μM were used alone or in combination with 5 μM ALS to treat the cells for 24 hours. At the end of treatment, the cells were trypsinized and centrifuged at 1,000× g for 5 minutes to pellet the cells. The cells were divided into two samples of equal volume and washed with PBS. For detection of apoptosis using Annexin V, each sample was resuspended in 1 mL of binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2; filtered through a 0.2 μm filter). An aliquot of cell suspension (195 μL) was mixed with 5 μL of Annexin-ATTO 647N (Enzo Life Sciences Inc; ALX-209-259-T100), and incubated for 10 minutes in the dark. The cells were washed once using PBS and resuspended in 190 μL of binding buffer; 10 μL of 20 μg/mL propidium iodide was then added, and the cells were subjected to apoptotic analysis using flow cytometry. For detection of autophagy, each sample was washed by resuspending the cell pellet in 1× assay buffer (Enzo Life Sciences Inc; ENZ-51031-K200) and collected by centrifugation. Cells were resuspended in 250 μL of phenol red-free culture medium (Invitrogen; #1294895) containing 5% fetal bovine serum, and 250 μL of diluted Cyto-ID green stain solution (Enzo Life Sciences Inc; #ENZ-51031-K200) was added to each sample and mixed well. Cells were incubated for 30 minutes at 37°C in the dark, collected by centrifugation, washed with 1× assay buffer, and resuspended in 500 μL of fresh 1× assay buffer. Cells were analyzed using the green (FL1) channel of a flow cytometer.
Confocal fluorescence microscopy
The cellular autophagy level was examined using a Cyto-ID autophagy detection kit according to the manufacturer’s instructions. The SKOV3 and OVCAR4 cells were seeded into an eight-well chamber slide at 30% confluence. After incubation overnight, the cells were treated with ALS 0.1, 1, and 5 μM for 24 hours. In separate experiments, cells were pretreated with the autophagy inducer SB203580 (10 μM), autophagy inhibitor wortmannin (10 μM), chloroquine (30 μM), bafilomycin A (10 μM), and the apoptosis inhibitor Z-VAD(OMe)-FMK (20 μM) for 1 hour, and then treated with ALS 5 μM. After incubation for 24 hours, the cells reached ~60% confluence, were washed three times with 1× assay buffer, and then fixed by paraformaldehyde. A 40 μL aliquot of assay buffer with 4′ 6-diamidino-2-phenylindole was used to stain DNA molecules in the cells. The slides were covered with glass coverslips and sealed with polish oil. Samples were observed using a TCS SP2 laser scanning confocal microscope (Leica, Wetzlar, Germany) at a 405/488 nm wavelength. Full paper
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