Annexin 5 and pi staining kit

Annexin and PI staining was performed by using Annexin V and PI staining kit (BD Bioscience) according to the manufacturer's instruction. Briefly, cell-cultured medium was collected. The mESCs were detached with a 0.05% Trypsin and 0.5 mM EDTA solution and the collected medium was added to the cell suspension, followed by the addition of soybean trypsin inhibitor (0.05 mg/ml). Next, 1 × 10⁵ cells were resuspended in 1 × binding buffer, immunostained with 5 μl of FITC Annexin V and 5 μl of PI, and incubated at 25 °C for 15 min in the dark. Analysis was performed by using flow cytometry (Beckman Coulter, Fullerton, CA, USA). From each sample, 5 × 10³ cells were obtained and analyzed by using CXP software (Beckman Coulter).

Apoptosis and necrosis of cells were measured with an Annexin V and PI staining kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. Briefly, the cells were detached with 0.05% trypsin/EDTA and 1 × 10⁵ cells were suspended with Annexin V binding buffer. The cells were stained with Annexin V and PI and incubated for 20 min at room temperature in the dark. The sample was read by flow cytometry and analyzed using CXP software (Beckman Coulter, Brea, CA, USA). Samples were gated to exclude debris (forward light scatter [FSC] area vs. side scatter area), and then any cell doublets were excluded using FSC area vs. FSC width analysis.

The necrotic cell death was evaluated with an Annexin V and PI staining kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. Briefly, single cell suspension was prepared with Annexin V binding buffer at 1 × 10⁵ cells and stained with Annexin V (25 μg/mL) and PI (125 ng/mL) for 15 min at room temperature in the dark. The sample was examined by flow cytometry and analyzed using CXP software (Beckman Coulter, Brea, CA).

Cells were synchronized in the G₀/G₁ phase by culture in serum-free media for 24 h before incubation of rVvpM The necrotic cell death was detected with an Annexin V and PI staining kit (BD Biosciences, Franklin Lakes, NJ) according to the manufacturer's instructions. Briefly, the cells were detached with 0.05% trypsin/EDTA and 1 × 10⁵ cells were resuspended with Annexin V binding buffer. And then the cells were stained with Annexin V (25 μg/ml) and PI (125 ng/mL), and incubated for 15 min at room temperature in the dark. The sample was read by flow cytometry and analyzed using CXP software (Beckman Coulter, Brea, CA). Samples were gated to exclude debris (forward light scatter [FSC] area vs. side scatter-area), and then any cell doublets were excluded using FSC-area vs. FSC-width analysis.

Cells were synchronized in the G₀/G₁ phase by culture in serum-free media for 24 h before incubation of rVvhA. The cell death of INT-407 cells was detected with an Annexin V and PI Staining Kit (BD Biosciences) according to the manufacturer's instructions. Briefly, the cells were detached with 0.05% trypsin/ethylenediaminetetraacetic acid (EDTA), and 1 × 10⁵ cells were resuspended with Annexin V-binding buffer (0.1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)/NaOH (pH 7.4), 1.4 M NaCl, 25 mM CaCl₂). And then the cells were stained with Annexin V (25 μg/ml) and PI (125 ng/ml) and incubated for 15 min at room temperature in the dark. The sample was read by flow cytometry and analyzed using the CXP software (Beckman Coulter, Brea, CA, USA).

S. aureus (strain RN6390) was maintained in tryptic soy broth (Sigma-Aldrich, St. Louis, MO, USA). Antibodies against caspases (caspase-3: Sc7148; caspase-9: Sc8355), cytochrome c (Sc7159), AIF (Sc5586), PARP-1 (Sc25780), Bax (Sc526) and Cox 4 (Sc292052) were purchased from Santa Cruz Biotechnology (Paso Robles, CA, USA). A mouse monoclonal anti-β-actin (A2228) antibody was purchased from Sigma-Aldrich. Secondary horseradish peroxidase-conjugated anti-mouse (170–6516) or anti-rabbit (170–6515) IgG antibodies were purchased from Bio-Rad (Hercules, CA, USA). Annexin V and PI staining kit, and JC-1 staining kits were purchased from BD Biosciences (San Jose, CA, USA). Caspase and PARP-1 inhibitors were purchased from R&D Biosciences (Minneapolis, MN, USA). ApopTag Fluorescein In situ Apoptosis Detection Kit was purchased from Millipore (Billerica, MA, USA).