Annexin 5 fluorescein isothiocyanate fitc

Viable cell numbers were counted based the Trypan blue exclusion (Corning Inc., Corning, NY, USA). Cell apoptosis was detected by staining with Annexin V-Fluorescein isothiocyanate (FITC) and 7-AAD (BioLegend). Briefly, 2 × 10⁵ primary mSMG cells were incubated with recombinant murine IL-22 (20 ng/mL) for 3 days. The cells were subsequently stained with Trypan blue solution for viable cell enumeration, or incubated with Annexin V-FITC plus 7-AAD (BioLegend) for apoptosis detection. The cells were then analyzed using a BD FACSAria III cell sorter/flow cytometer and the Flowjo software to determine the percentage of cells stained positive for Annexin V and/or 7-AAD.

To examine cell death resultant from carboplatin (0.025 mg/mL; IPO’s Pharmacy) exposure with and without cysteine (0.402 mM; 102839, Merck) or the carboplatin cysteinyl-S-conjugate (the metabolite generated upon the catabolism of GSH–carboplatin adduct) [51 (link)], cells (2 × 10⁵ cells/mL) were seeded in 24-well (500 µL/well) plates incubated for 16 h. In the case of the SeChry (19 µM, a concentration above EC₅₀; see Figure S1) effect on cell death, an incubation of 48 h with SeChry was performed, and, in the last 24 h, cysteine and/or carboplatin were added. After experimental conditions, the supernatants were collected, and adherent cells were harvested with 0.05% trypsin–EDTA. Cells in the supernatant and trypsinized cells were joined and centrifuged at 255× g for 2 min. Cells were stained with 0.5 μL annexin V–fluorescein isothiocyanate (FITC) (640906, BioLegend, San Diego, CA, USA), in annexin V binding buffer 1×, and incubated at RT, in dark for 15 min. Samples were resuspended in 200 μL PBS (1×) with0.1% BSA and centrifuged at 255× g for 2 min. Cells were resuspended in 200 μL of annexin V binding buffer 1×, and 2.5 μL of propidium iodide (PI, 50 μg/mL; P4170, Sigma-Aldrich) was added 5 min prior to analysis. Afterward, samples were analyzed by flow cytometry (FACScalibur, Becton Dickinson). Data were analyzed using FlowJo 8.7 software (https://www.flowjo.com).

Adherent macrophages (2 × 10⁶) in 6-well microplates were exposed for 1 h to purified LtxA (1 µg/mL) in the absence or presence of different concentrations of cranberry PACs at 37 °C in a 5% CO₂ atmosphere. The macrophages were washed twice with ice-cold PBS and were detached by adding Accutase^® (1 mL; Innovative Cell Technologies Inc., San Diego, CA, USA) for 7 min (37 °C). The viability of macrophages was determined by staining with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) (BioLegend, San Diego, CA, USA) for 15 min. To assess caspase-1 activation, the macrophages were stained with AM-VAD-FMK reagent, a fluorochrome inhibitor of caspase-1 (FLICA), according to the manufacturer’s protocol (Thermo Fisher Scientific). Cells were washed to remove unbound reagent. A total of 20,000 cells were analyzed using a Cytomics FC 500 flow cytometer (Beckman Coulter Inc., Indianapolis, IN, USA). Data were analyzed using CXP software (Beckman Coulter Inc., Indianapolis, IN, USA) and Flowing software 2.51 (Perttu Terho, Center for Biotechnology, Turku, Finland)