Caspase 8 activity assay kit

Caspase 8 activity was measured using the caspase 8 activity assay kit (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer’s instruction. Briefly, cells were collected by trypsinization and lysed with lysis buffer. The protein concentrations in the lysates were quantitated with a Bradford assay kit (Bio-Rad). The lysates were mixed with the caspase 8 substrate (Ac-IETD-pNA) in a 96-well plate and then incubated at 37 °C for 30–120 min. The absorbance was measured at 405 nm and used to calculate caspase 8 activity. The relative activity of caspase 8 was calculated by normalizing the caspase 8 activity of each group with that of the normal control group.

The cells were homogenized in lysis buffer and centrifuged at 16000 × g for 15 min at 4 °C. The supernatants were collected to detect the activity of caspase-8 with a caspase-8 activity assay kit (C1152, Beyotime) according to the manufacturer’s instructions. The activity of caspase-8 was evaluated according to the absorbance measured at 405 nm. Then the results were normalized to the protein concentrations and are shown as percentages of the specific value.

HEK293T cells were treated with DMSO, Z-DEVD-FMK, Z-IETD-FMK, and Z-LEHD-FMK (20 mM) for 2 h. The culture medium was replaced after 2 h. Caspase activity was measured after 16 h using a Caspase 3 Activity Assay Kit, a Caspase 8 Activity Assay Kit, or a Caspase 9 Activity Assay Kit (Beyotime, Shanghai, China).

McCoy's 5A medium, fetal bovine serum (FBS), penicillin-streptomycin and trypsin-EDTA were obtained from Gibco (Grand Island, NY, USA). NAC, Ac-DEVD-CHO, DAPI and AO&EB, DCFH-DA, JC-1 stain kit, Cell and Tissue mitochondria isolation kits, Caspase-8 activity assay kit and BCA protein assay kit were obtained from Beyotime Biotech (Jiangsu, China). Nuclear-Cytosol kit was obtained from Applygen Technologies Inc (Beijing, China). Annexin V-FITC/PI assay kit, ROS and TNF-α ELISA kits were purchased from KeyGEN Biotech (Nanjing, China). Antibodies against Bax (^#2772), Bcl-2 (^#4223), Cytochrome c (^#11940), Caspase-3 (^#9662), cleaved Caspase-3 (^#9661), Caspase-8 (^#4790), cleaved Caspase-8 (^#9496), Caspase-9 (^#9508), NF-κB (p65, ^#8242) and Histone H3 (^#3638) were purchased from Cell Signalling Technology (Danvers, MA, USA). Anti-β-actin and anti-GAPDH antibodies, anti-mouse and anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies, 5-Fluorouracil (5-FU), MTT and standard sugar were purchased from Sigma (St. Louis, MO, USA). All other chemicals used were of analytical grade.

Caspase activity was measured with Caspase-8 Activity Assay Kit, Caspase-9 Activity Assay Kit, and Caspase-3 Activity Assay Kit (Beyotime) according to the manufacturer’s instruction. Cells were lysed after exposed to gradient concentrations of SFE for 24 h and 48 h, respectively. Substrates were diluted to 0, 10, 20, 50, 100, and 200 µM as standards. Cell lysates were mixed with 10 µL of 2 mM substrate and reaction buffer to a total volume of 100 µL, and incubated at 37 °C for 2 h. Absorbance of samples and standards was measured with microplate reader (Bio-Rad) at 405 nm.

Caspase-3, -8, and -9 activities were quantified using the Caspase 3 Activity Assay Kit (C1116), Caspase 8 Activity Assay Kit (C1152), and Caspase 9 Activity Assay Kit (C1158, all from Beyotime), according to the manufacturer’s protocols. Briefly, at 2 dpi, cells cultured in 6-well plates were collected and centrifuged at 600 × g at 4°C for 5 min, and then resuspended in ice-cold cell lysis buffer on ice for 15 min. Cell lysates were again centrifuged, at maximum speed and 4°C for 10 min, before the supernatant was collected. After the reaction buffer and corresponding caspase substrate were added, the mixture was incubated in a water bath at 37°C for 60 min. The substrates of caspase-3, -8, and -9 were Ac-DEVD-pNA, Ac-IETD-pNA, and Ac-LEHD-pNA, respectively. Absorption of the preparation at 405 nm directly correlated with the concentration of pNA and thus allowed us to calculate the activity of the respective enzyme. Activities are expressed as fold change over the activity detected in mock-infected cells. As for caspase-8 activity assay, a positive control was set via the TNF-α treatment. NA cells were treated with 100 ng/mL TNF-α for 6 h and then collected for caspase-8 assay.