Trypan blue staining cell viability assay kit

HaCaT cells were treated with or without 50 mJ/cm² UVB radiation; then, the cells were cultured in the presence or absence of 100 mM trehalose for 12 or 24 hours. Subsequently, the cells were digested and stained using a Trypan blue staining cell viability assay kit (Beyotime Biotechnology) to assess cell viability in a blood cell counting chamber according to the manufacturer’s instructions47 (link). The percentage of surviving cells = (total number of cells – number of trypan blue positive cells)/total number of cells.

The spleen and head kidney tissues, and peripheral blood samples were collected from zebrafish with designated treatments in ice-cold D-Hank’s with heparin sodium (10 U/ml, Sigma-Aldrich). Whole-blood cell suspensions were obtained with heparinized capillary tubes, and single-cell suspensions of spleens and head kidneys were prepared by gently teasing the tissues through an 80-mm nylon mesh filter. Leukocytes were enriched from the cell suspensions by Ficoll-Hypaque (1.080 g/ml) centrifuging at 2,500 rpm for 25 min at room temperature, collected from the interface layer, and washed thrice with ice-cold D-Hank’s at 350 g for 10 min at 4°C. For primary cell culture, the isolated leukocytes or sorted lymphocytes were seeded into 24-wells plate (Corning) with Leibovitz’s L-15 Medium (L-15, Gibco) including 10% (v/v) FBS, 100 U/ml of penicillin and 100 μg/ml of streptomycin at 28°C in 5% CO₂ for indicated time. Cell viability was assayed with Trypan Blue (Trypan Blue Staining Cell Viability Assay Kit, Beyotime) and the leukocyte samples with cell viability ≥ 95% were used in the next experiments.

Monoclonal antibodies against Bcl-2, Bax, P53, P21, Mcl-1, and GAPDH were purchased from Santa Cruz (Santa Cruz, CA, USA). Antibodies against CytC, Fas, FasL, and TRAIL were purchased from Abcam (Cambridge, MA, USA). Mouse anti-γ-H2AX antibody was from Millipore (Billerica, MA, USA). DMEM medium and FBS were purchased from Gibco BRL (Grand Island, NY, USA). FITC annexin V Apopotosis Detection Kit was from BD Biosciences (San Jose, CA, USA). Caspase-Glo 3/7, 8, and 9 assay kits and Plasmid extraction kit were purchased from Promega (Madison, WI, USA). N-acetyl-L-cysteine (NAC) and Reactive Oxygen Species Assay Kit were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lipofectamine^TM 2000, Dichloro-dihydro-fluorescein diacetate (DCFH-DA), MitoSOX^TM Red, MitoTracker Deep Red FM, MitoTracker Green FM and Hoechst 33342 were from Invitrogen (Carlsbad, CA, USA). Trypan Blue Staining Cell Viability Assay Kit was from Beyotime Institute of Biotechnology (Jiangsu, China). Tetramethylrhodamine methyl ester (TMRM) was from Life Technologies (Carlsbad, CA, USA). Z-LEHD-FMK, Z-IETD-FMK, Z-DEVD-FMK were from Calbiochem (San Diego, CA, USA). Colorimetric TUNEL Apoptosis Assay Kit was from Beyotime (Shanghai, China). Mitochondria/Cytosol Fractionation Kit was from BioVision (San Francisco, CA, USA). DL-2000 DNA Marker was from TaKaRa Biotechnology (Dalian, China).