Vybrant apoptosis assay kit 3

VitK3, N-ethylmaleimide (NEM), N-acetyl-L-cysteine (NAC), dimethyl sulfoxide (DMSO), dithiothreitol (DTT), H₂O₂ and trichloroacetic acid (TCA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Vinblastine, taxol, doxorubicin, daunorubicin, actinomycin D and 5-fluorouracil were from Enzo Life Sciences (Villeurbanne, France). Hygromycin B, 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (carboxy-H₂DCFDA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), crystal violet, Vybrant Apoptosis Assay Kit #3, propidium iodide (PI), tetramethylrhodamine methyl ester (TMRM), 4-acetamido-4’-maleimidylstilbene-2,2’-disulfonic acid (AMS) were from Life Technologies (Eugene, OR, USA). The following antibodies were used: anti-PRX1 (AbFrontier, Seoul, Korea); anti-PRX3 (Abcam, Cambridge, MA, USA); anti-TRX2 (R&D Systems, Minneapolis, MN, USA); anti-NQO2 (Proteintech, Chicago, IL, USA); anti-GFP (Life Technologies); anti-β-actin (Santa Cruz Biotechnology); anti-α-tubulin (Sigma-Aldrich) and anti-TRX1 (Cell Signaling Technology, Danvers, MA, USA). ON-TARGETplus SMARTpool small interfering RNA (siRNA) targeting PRX1 (siPrx1), and non-targeting pool control siRNA (siCon) were from Dharmacon (Lafayette, CO, USA). Mammalian expression vectors encoding HyPer targeted to cytosol, nucleus and mitochondria were purchased from Evrogen (Moscow, Russia).

FITC-annexin V and PI staining was performed using Vybrant Apoptosis Assay Kit #3 (Life Technologies, Carlsbad, CA, USA) following the manufacturer’s directions. After being treated, floating cells were harvested with medium and attached cells were dissociated with EDTA-trypsin solution. Cells were collected by centrifugation at 1000 r.p.m. for 5 min. Cells were centrifuged and washed twice with phosphate-buffered saline (PBS), and the pellets were suspended in 100 μl binding buffer containing 10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl (pH 7.4), and incubated with 5 μl of FITC-annexin V and 1 μl of 100 μg/ml PI solution for 15 min at room temperature. Thereafter, 400 μl of binding buffer was added, mixed gently, and kept on ice. Stained cells were analyzed by FACSCalibur (Becton Dickinson, Mountain View, CA, USA). Data were analyzed by Cell Quest software (Becton Dickinson).

FITC-annexin V and PI staining was performed using Vybrant Apoptosis Assay Kit#3 (Life Technologies, Carlsbad, CA, USA) following the manufacturer’s instructions. After being treated, floating cells were harvested with medium and attached cells were dissociated with EDTA-trypsin solution. Cells were collected by centrifugation at 1,000 rpm for 5 min. Cells were centrifuged and washed twice with phosphate-buffered saline (PBS), and the pellets were suspended in 100 μl binding buffer containing 10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl (pH 7.4) and incubated with 5 μl of FITC-annexin V and 1 μl of 100 μg/ml PI solution for 15 min at room temperature. Thereafter, 400 μl of binding buffer was added, mixed gently and kept on ice. Stained cells were analyzed by FACSCalibur (Becton Dickinson, Mountain View, CA, USA). Data were analyzed by Cell Quest software (Becton Dickinson).
Regarding acridine orange staining, cells floating and attached cells were harvested and collected by centrifugation at 1,000 rpm for 5 min. Acridine orange was added at a final concentration of 1 μg/ml and incubated for 15 min. The emission of red (564–606 nm) fluorescence was measured with a FACSCalibur.