Annexin 5 pi

Lung cancer cells were cultured in 6-well plates with ciclesonide (20 μM). Apoptotic cells were detected by Annexin V/PI staining according to the manufacturer’s instructions (BD, San Jose, CA, USA). The samples were analyzed by Accuri C6 (BD, San Jose, CA, USA). Cancer cells were treated with 20 μM ciclesonide for 1 day, and then the cells were incubated with Hoechst 33258 (10 mg/mL) solution for 20 min at 37 °C. The cells were observed using a fluorescence microscope (Lionheart FX, Biotek, Winooski, VT, USA).

HCC cells were cultured in six-well plates and treated as indicated. After 24 h, the cells were centrifuged, washed twice with PBS, and mixed in 100 μl of 1× binding buffer (10mM HEPES/NaOH, PH7.4, 140mM NaCl, 2.5mM CaCl₂). After culturing for 15 min at room temperature in Annexin-V/PI (BD Biosciences, San Jose, CA) double staining liquid, the cells were examined by flow cytometry (Cytomics FC500; Beckman Coulter, Fullerton, CA). The percentage of apoptotic cells was calculated using ModFitLT software.

Cells in the log phase were seeded onto a 6-well culture plate at a density of 1×10⁵ cells per well, incubated at 37°C in a CO₂ incubator, and allowed to attach overnight. After treatment with Au-NPs (control, 5 nm, 10 nm, 20 nm, and 40 nm) for 48 h, apoptosis and necrosis were analyzed with the Annexin V-PI (BD Biosciences) apoptosis detection kit following the manufacturer’s instructions. The samples were analyzed using a BD FACS CantoII instrument (BD Biosciences).

HCC cells were cultured in six-well plates and treated as indicated. After 24 h, the cells were centrifuged, washed twice with PBS, and mixed in 100 μl of 1× binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂). After culturing for 15 min at room temperature in Annexin-V/PI (BD Biosciences) double staining liquid, the cells were examined in a flow cytometer (Cytomics FC500). The percentage of apoptotic cells was calculated using ModFitLT software.

Induction of programmed cell death (apoptosis) was tested using the FACS Canto (BD‐Biosciences, Franklin Lakes, NJ, USA). 2.0 × 10⁵ BT‐20 cells were harvested and analysed at day 7 and day 8 post‐infection. Staining with Annexin V/PI (BD‐Biosciences) was performed for 30 min at 37 °C. Cells were washed with PBS and analysed by flow‐cytometry. Unstained cells were used as controls to check for auto‐fluorescence and channel spillover. Dead cells and cellular debris were excluded from the analysis (SSC/FSC; see Fig. S4A). Annexin V‐positive cells were assumed to be early apoptotic cells and percentages of this population were calculated using the flowjo software (BD‐Biosciences). For each day, n = 3 biological replicates were analysed.