Annexin 5 fitc pi apoptosis assay kit

We used RPMI1640 medium (HyClone, American), trypsin, a cell counting kit-8 (CCK-8), an annexin V-FITC/PI apoptosis assay kit, and 0.1% diethylpyrocarbonate (DEPC)-water (all purchased from Beijing Solarbio Science and Technology Co., Ltd.). We also used fetal calf serum (Sijiqing Co. Ltd. Lot 11011-8611), TRIzol (Ambion [American]), chloroform, isopropanol, and absolute ethyl alcohol (all from Tianjin Kemiou Chemical Reagent Co. Ltd.). The RNA extraction kit (lot TCF-514123) was obtained from Proteintech Group Inc. (Wuhan, China). The HiFiScript gDNARemoval cDNA Synthesis Kit (lot CW2582M) was obtained from Jiangsu Cowin Biotech Co., Ltd. ChemoHS qPCR Mix (None ROX) (lot MQ00101S) was obtained from Monad Biotech Co., Ltd. (Wuhan, China).

The in vitro cell modal assay was performed according to the method described previously [26], using human rectal cancer epithelial cell line Caco-2 (ATCC number: HTB-37™, Stem Cell Bank of Chinese Academy of Sciences, Shanghai, China). Briefly, Caco-2 cells (5 × 10⁴ cells/mL per well) were cultured in Dulbecco’s modified eagle medium (DMEM, Gibco, USA) at 37 °C with 5% CO₂ for 24 h, and then washed three times with 0.1 M PBS (pH 7.2–7.4, Sangon, China). Each of V. parahaemolyticus strains was grown in TSB medium (pH 8.5, 3% NaCl) at 37 °C to mid-LGP, and then harvested, washed, and resuspended in DMEM medium without phenol red. A 100 μL/well of bacterial suspension (OD_490 nm of about 0.2 ± 0.02), and 10 μL/well of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonate)-2h-tetrazolium monosodium salt (CCK-8, Sigma–Aldrich, USA) were added into Caco-2 cells per well, and then incubated at 37 °C for 4 h. Caco-2 cell viability and apoptosis were examined using Annexin-V-FITC/PI Apoptosis Assay Kit (Solarbio, China) and BD FACSVerse™ flow cytometer (Becton, Dickinson and Company, USA) according to the instructions of the manufacturers (Yang et al. 2020 (link)).