Caspase 3 assay kit ab39401

Cell death was determined by PI staining. Cells were exposed to normoxia or hypoxia for 4 or 72 h. After dissociation, cells were collected and centrifuged for 5 min at 300 × g. The pellet was washed once with PBS and resuspended in FACS buffer (PBS, 2% FBS, 1 mM EDTA, 0.1% sodium azide) containing 0.05 μg/ml PI. Cells were analyzed using FACSVerse instrument (BD, Franklin Lakes, USA) and data analyzed with FlowJo software.
Caspase-3 activity was measured with Caspase-3 Assay Kit ab39401 (Abcam) following manufacturer’s instruction.
All experiments were performed in triplicate.

Curcumin, ABTS (2,2′-azinobis-3-ethylbenzothiazolin-6-sulfonic acid), and DPPH (1,1′-diphenyl-2-picryhydrazyl) were obtained from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). β-CD (chemical purity) was purchased from Shangdong Xinda Biotechnology Co., Ltd. (Zibo, China). Epichlorohydrin (EP) and ethylene glycol were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). RPMI 1640 medium were purchased from Hyclone Company (South Logan, UT, USA). Fetal bovine serum (FBS) and the antibiotic mixture (penicillin-streptomycin) were purchased from Gibco Company (Grand Island, NY, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Company (Kumamoto, Japan). Caspase 3 assay kit (ab39401), Caspase 8 assay kit (ab39700), and Caspase 9 assay kit (ab65608) were purchased from Abcam Company (Cambridge, UK). Annexin V/PI apoptosis kit were purchased from MultiScience Company (Hangzhou, China). Sodium hydroxide and other reagents were of analytical grade and were purchased from Nanjing Chemical Reagent Co. Ltd. (Nanjing, China). Double distilled and sterilized water was used to prepare all solutions.