Luminescent caspase glo 3 7 assay kit

DEN, 2-AAF, 5,5′-dithiobis-(2-nitrobenzoic acid), thiobarbituric acid, Folin’s reagent, pyrogallol, SOD enzyme, H₂O₂, and bovine albumin were obtained from Sigma Chemical Co. (St. Louis, MO). Primary antibodies of Ki-67, COX-2 (Clone SP 21), iNOS (Ab-1), and NF-kB-P65 (Rel A, Ab-1) were purchased from Thermo Fisher Scientific, Anatomical Pathology, Fremont, USA (1:100 dilutions). GST-p form was obtained from Medical and Biological laboratories Co., Tokyo, Japan (1:1,000 dilution). M30 CytoDeath was purchased from Enzo life Science, USA. Anti-CD68 (ED1), -CD163 (ED-2) (1:300 dilution), and -phosphorylated form of tumor necrosis factor alpha receptor 1 (p-TNFR) (1:200 dilution) antibodies were obtained from Santa Cruz, CA, USA. Safranal (W338907 Aldrich) was obtained from Sigma-Aldrich, USA. CellTiter-Glo luminescent Cell viability assay kit and Caspase-Glo 3/7 luminescent assay kit were obtained from Promega (Woods Hollow. Rd., Madison Wisconsin, USA). HDAC Colorimetric Assay Kit and Human IL-8 ELISA Kit (EZHIL8) were obtained from the Millipore Corporation (28820 Single Oak Drive, Temecula, CA 92590, USA) and the TNF-α ELISA kit came from R&D Systems (Minnesota, USA).

Cells were cultured for 48h in the presence of indicated concentrations of compounds, harvested and washed, and incubated with Annexin V-FITC (BD Biosciences) and Propidium Iodide for 15min at room temperature in the dark. Apoptosis was measured by BD LSR II flow cytometry (BD Biosciences).
Caspase-3/7 activity assay was performed on the HMCLs using Caspase-Glo 3/7 luminescent assay kit according to manufacturer’s instructions (Promega Madison, WI). Briefly, 1×10⁶/ml cells were cultured in 6 well plates and treated with either CLF alone and/or combination for indicated times. After harvesting, cells were washed twice with cold PBS and resuspended with Caspase-Glo 3/7 reagents. Reactions were incubated for 1h at 37°C, and luminescence was determined using Synergy 2 Microplate Reader (Biotek). Cell death by apoptosis was also measured by immunoblotting analysis.

HepG2 cells were seeded in 96-well plates at the density of 10,000 cells/well and grown in 100 µl of complete growth medium. Complete growth medium was replaced by serum-free medium after cells were allowed to attach for 24 h, after which cells were incubated for at least 12 h. Cells were incubated for 48 h after treatment with various concentrations of safranal (1, 0.7, and 0.5 mM) prepared from 20 mM stock solution. After the incubation period, caspase-3 and -7 activities were measured using Caspase-Glo 3/7 luminescent assay kit according to manufacturer’s instruction (Promega G8091). Luminescent signal was detected using GloMax Discover System (Promega). The induction of DNA double-strand breaks (DSBs) was assessed by measuring phosphorylation of the histone H2X using western blotting.

To induce apoptosis, we used a HR/SS protocol as described in Figure 1 Scheme A. After culture in regular culture conditions for 24 hrs, the cells were serum‐starved with medium containing 0.1% FBS under hypoxia for 24 hrs (1% O₂). HP was added at the beginning of reoxygenation (21% O₂). At the end of the experimental procedure, the same volume (50 μl from each well) of conditioned media was collected and used for caspase‐3/7 activity assay using a luminescent Caspase‐Glo 3/7 Assay kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. At the same time, the cells were lysed and used for the detection of fragmented nucleosomes using a Cell Death Detection kit (Roche, Indianapolis, IN, USA) as described 9.