Caspase 3 7 activity detection kit

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), 4′,6-diamidino-2-phenylindole (DAPI), penicillin–streptomycin, and 0.25% (w/v) trypsin containing 1 mM ethylenediaminetetraacetic acid were purchased from Invitrogen (Carlsbad, CA), and 6-OHDA, 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), vitamin C, dimethyl sulfoxide (DMSO), Akt inhibitor IV, MEK inhibitor (PD 98059), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO). Nerve growth factor (NGF) was obtained from R&D Systems (Minneapolis, MN, USA). A total antioxidant capacity assay kit was purchased from Abcam (Cambridge, UK). A lactate dehydrogenase (LDH) cytotoxicity assay kit was purchased from Cayman Chemical (Ann Arbor, MI). A Caspase 3/7 activity detection kit was obtained from Promega (Madison, USA). Antibodies for western blotting were purchased from Cell Signaling Technology (Danvers, MA). All chemicals were dissolved in appropriate solvents and stored at −20°C before use to maintain their chemical stability.

After 6 h of culture in Transwell (5 × 10⁴ cells), the cells were washed with PBS and treated with hypotonic buffer (10 mm tris‐HCl, 10 mm KCl, 1.5 mm MgCl₂, pH 7.5) for 10 min. Then the chambers were inserted into 1.5 mL Eppendorf tubes and centrifuged for 10 min with 12 000 × g and the supernatants were collected. For cells cultured in dishes, the cells were resuspended in hypotonic buffer, after trypsinizing and PBS washing, for 10 min and broken by grinding with a grinder (Bio Vision). Then the cell lysates were centrifuged for 10 min with 12 000 × g and the supernatants were collected. Finally, the activities of caspase‐3/7 in the collected supernatants were detected with the caspase‐3/7 activity detection kit of Promega following the manufacture's instruction.