Annexinv pi apoptosis assay kit

MTT powder (1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan), RIPA buffer, TRIZOL reagent, CoCl₂, DCF-DA, and Protoporphyrin IX (SnPP) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). AnnexinV/PI apoptosis assay kit was obtained from BD Biosciences (San Diego, CA, USA). Antibodies against Caspase3, Caspase7, Caspase8, Caspase9, HIF1α, p53, VEGF, GLUT1, SOD, CAT, HO-1, and LaminB were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Bcl-2, anti-β-actin, and anti-Nrf2 antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The RT PreMix kit was purchased from Enzynomics (Daejeon, Korea). SYBR Premix Ex Taq was obtained from Takara (Shiga, Japan). Nuclear and Cytoplasmic Extraction Reagents Kit (NE-PER) and ECL Western blot detection reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Cells were digested by trypsin, resuspended in 4 mL PBS, and centrifuged at 1000 rpm for 12 min. Then, cells were fixed with 75% precooled ethanol at 4°C overnight, followed by staining in 1 mL PI. Cell cycle was detected using flow cytometry (BD Biosciences, San Jose, CA), and proliferation index (PI, G₂/M + S) in each group was counted and compared. For cell apoptosis detection, cells were digested by EDTA-free trypsin, resuspended in 6 mL PBS and centrifuged at 1000 rpm for 6 min twice, and then resuspended again in binding buffer. Annexin V/PI apoptosis assay kit (BD Pharmingen, Franklin Lakes, USA) was applied to detect cell apoptosis within 15 min in dark. flow cytometry was employed to calculate the apoptotic rate. In this study, the number of apoptotic cells was defined as the early apoptotic cells (Q2: annexin V+/PI− staining) and the late apoptotic cells (Q4: annexin V+/PI+ staining).

Annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) staining by flow cytometry illustrated the oncolytic RSV-A2-induced apoptosis of TC-1 cancer cell lines. Using a commercial Annexin V /PI Apoptosis Assay Kit (BD Biosciences, USA), the impact of RSV-A2 on apoptosis in TC-1 tumor cell lines was analyzed. Briefly, each well of a six-well plate was seeded with 5 × 10⁵ cells. Then, TC-1 cell lines were treated with RSV-A2 virus at MOIs of 1, 5, 10, and 15 for one hour, and the medium was replaced with DMEM with 1% pen/strep and 1% FBS for seventy-two hours. Non-infected cell lines were regarded as a control group. The TC-1 cancer cells were trypsinized and exposed to the DMEM to eliminate the effect of the trypsin. Next, 100ml of the binding buffer was appropriately mixed with 5ml of propidium iodide reagent and 5ml of FITC-conjugated anti-annexinV/PI labeling antibody (BD Biosciences, USA). After 15 min of incubation in the dark and at room temperature, the apoptotic percentage of the cells was determined. The average proportion of annexin V-stained cells versus the control group was compared in flow cytometry annexin V staining. Each test was conducted three times.

Annexin V/PI apoptosis assay kit was used according to the protocol provided by the manufacturer (BD Biosciences Pharmingen). The HCC cells were harvested and washed twice with cold PBS, then resuspended in binding buffer at a concentration of 1×10⁶ cells/ml. Fluorescein isothiocyanate (FITC)-conjugated annexin antibody (5 µl) and prodium iodide (PI) solution (5 µl) were added into prepared cell suspension, and the mixtures were incubated for 30 minutes at room temperature in dark place. The cell suspension was analyzed using the flow cytometry.

Cell death was detected using the SYTOX dead cell stain sampler kit (#S34862, Invitrogen) and the Annexin V-PI apoptosis assay kit (#556547, BD). Briefly, the indicated cells were plated in 12-well plates and treated with different concentrations of cytotoxic compounds for the indicated times. Then, SYTOX stain was added to the cell supernatants, and the cells were incubated at room temperature for 10 min, followed by observation and imaging using a fluorescence microscope. The Annexin V-PI apoptosis assay was quantified using flow cytometry.

AnnexinV and caspase3/7 staining reagents for IncuCyte^® cell imaging system were obtained from Essen bio (Ann Arbor, MI, USA). AnnexinV/PI apoptosis assay kit was purchased from BD Biosciences (San Diego, CA, USA). Anti-CD3 and anti-CD28 for stimulation were obtained from Bioxcell (West Lebanon, NH, USA). Salinosporamide A, MTT (1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan) powder, PMA (Phorbol 12-myristate 13-acetate), and A23187 was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Carboxyfluorescein succinimidyl ester (CFSE) dye, ECL Western blotting detection reagents, and NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit were obtained from Thermo Fisher Scientific (Waltham, MA, USA). PVDF membrane was obtained from Bio-Rad (Hercules, CA, USA). Anti-bcl-2, anti-β-actin, anti-cyclin A, anti-cyclin D1, and cyclin E antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-caspase3, anti-caspase7, anti-p65, anti-PARP, anti-IκBα, anti-ERK, anti-p38, anti-JNK, anti-phosphorylated IκBα (S32), anti-phosphorylated ERK (T202/Y204), anti-phosphorylated p38 (T180/Y182), and anti-phosphorylated JNK (T183/Y185) antibodies were obtained from cell signaling technology (Danvers, MA, USA).