Cell counting kit

The cell numbers were determined using an automatic cell counter (Nexcelom, Cellometer Mini, USA), and the trypan blue exclusion method was used for cell viability detection. In addition, the fourth passage cells were harvested for cell proliferation, apoptosis, growth curve, and cell cycle assays as a complementary experiment to decipher the viability of cells. The 5-ethynyl-2′-deoxyuridine (EDU, RiboBio Co., China) and Cell Counting Kit (Beyotime, China) were performed according to the manufacturer’s instruction, then the proliferation rate and growth curve were calculated or drawn, respectively. The apoptosis assay was performed with the Annexin V-FITC Apoptosis Detection Kit (Vazyme, China). The BD Cycletest™ Plus DNA Kit (BD, USA) was used to determine the cell cycle. Before releasing the final cell products, the cell count and viability assay also were performed and the viability must be over 85%. A tumor cell line (murine melanoma B16F10 cell) was cultured in an independent incubator as a positive cell control in all the above experiments because of its rapid and stable growth rate. In cell viability and apoptosis assays, a dose of 800 μM H₂O₂ was added to HUCMSC culture medium to induce cell apoptosis as positive controls to ensure the reliability of the experiments.

RNAiso plus 9109 Q (TaKaRa Tokyo, Japan); Hifair^® III 1st Strand cDNA Synthesis Kit (gDNA digester plus)11139 ES 10 (YEASEN, Shanghai, China); Hieff UNICON^® Universal Blue Qpcr SYBR Green Master Mix 11184 ES 03 (YEASEN); Recombinant flagellin protein derivative CBLB502 (InvivoGen, Toulouse, France); TIRF-specific antagonist Pepinh-TRIFTFA (MCE, Sichuan, China); EMEM culture medium (ATCC, Manassas, VA, USA); DMSO (Sigma-Aldrich, St. Louis, MO, USA); Fetal bovine serum (Gibco, Waltham, MA, USA); Plasmid mini extraction kit (Omega, Norcross, GA, USA); Plasmid maxi extraction kit (Omega, Norcross, GA, USA); EcoRI (TaKaRa, Japan); BamHI (TaKaRa); RNAisoplus (TaKaRa); TLR5 antibody (rabbit source; Proteintech, Rosemont, IL, USA); TRIF antibody (rabbit source, Affinity, USA); P-ERK1/2 antibody (rabbit source; Abcam, Waltham, MA, USA); CD80 (B7-1) Monoclonal Antibody, PE (eBioscience, San Diego, CA, USA); CD86 (B7-2) Monoclonal Antibody, PE (eBioscience, San Diego, CA, USA); MHC Class I (H-2Kb) monoclonal antibody, PE (eBioscience, San Diego, CA, USA); MHC Class II (I-A/I-E) monoclonal antibody, PE (eBioscience); Cell Counting Kit (Beyotime Biotechnology Co., Ltd., Haimen, China); rat interleukin 4 (IL-4) ELISA (Jiangsu Jingmei Bioengineering Co., Ltd., Guangzhou City, China); rat interleukin 12 (IL-12) ELISA kit (Jiangsu Jingmei Bioengineering Co., Ltd., Guangzhou City, China).

Cell viability was tested using a Cell Counting Kit (CCK8, Beyotime, China). The same density of cells was seeded into a 96‐well plate and cultured for 24 h with or without stimulators (DOX 1 µg mL⁻¹, Tg 10 nM) treated for indicated time points. Then, 10 µL of CCK8 reagent was added, maintained at 37 °C for 1 h. The optical density was then measured at 450 nm using an automatic i‐control microplate reader (Tecan Life Sciences, Switzerland).

Cell viability was assessed by the Cell Counting Kit (Beyotime, China). In 96-well culture dishes, 1 × 10⁵ macrophages in each well and cell were pretreated with 60% H₂ for 24 h and then stimulated with LPS (200 ng/ml) or PBS for another 24 h. Then, a 20 μl CCK-8 solution was added to each well and incubated at 37 °C for another 4 h. The cell viability was measured by a microplate spectrophotometer (Thermo, USA) at OD450 nm.