Caspase 3 activity assay kit

The effects of FFH on in vitro caspase-3 activity were determined using a caspase-3 activity assay kit (Enzo Life Sciences, Farmingdale, NY, USA). Briefly, PC-3 cells (1×106 per well) were incubated at 37°C in 5% CO₂ for 18 h and then with various concentrations of FFH for 24 h. Collected cells were lysed and cell lysates were quantified using the BCA method. Cell lysates (100 μg), fluorogenic peptide substrate (200 μM), and DEVD-p-nitroaniline (pNA; final concentration 200 μM) were then added in 96-well plate and incubated at 37°C in 5% CO₂ for 1 h. Absorbance was measured at 405 nm using a spectrophotometer (Tecan, Infinite® M200, Switzerland).[28 (link)]

Thiazolyl blue tetrazolium bromide (MTT), 2″,7″-dichlorodihydro-fluorescein diacetate (DCFH-DA) and other reagents were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Tris was purchased from Duchefa Biochemie (BH Haarlem, The Netherlands), dimethyl sulfoxide (DMSO) from Junsei Chemical Co. (Tokyo, Japan). Fetal bovine serum (FBS) and penicillin-streptomycin (P/S) were purchased from Gibco (Los Angeles, CA, USA). Dulbecco's Modified Eagle's Medium (DMEM) was purchased from Welgene Inc. (Gyeongsangbuk-do, Korea). BCA reagent and albumin standard were from Thermo Scientific (Waltham, MA, USA). Primary Bcl-2 antibody was purchased from Oncogene (Bracknell, England). Bax and PAPR antibodies were obtained from Cell Signaling (Danvers, MA, USA) and cytochrome c, caspase-9, and β-actin antibodies from Santa Cruz (Paso Robles, CA, USA). Caspase-3 antibody was obtained from EMD Millipore (Billerica, MA, USA). Secondary antibodies goat anti-rabbit IgG, pAb, goat anti-mouse IgG, pAb, and caspase-3 activity assay kit were obtained from Enzo Life Sciences (Farmingdale, NY, USA). West-Q chemiluminescent substrate was purchased from GenDEPOT (Katy, TX, USA). The solvents used were of HPLC grade, unless stated otherwise.