Caspglow fluorescein active caspase 9 staining kit

CT26 and HT29 cells were seeded in a 12-well plate (2 × 10⁵ cells/well) overnight and treated with 0, 75, or 100 μM magnolol for 24 h. Cells were then collected by 2000 rpm centrifugation and stained with 100 μL FACS buffer (0.5% FBS in phosphate-buffered saline) containing 1 μL cleaved caspase-3 (CaspGLOW™ Fluorescein Active Caspase-3 Staining Kit, BioVision), cleaved caspase-8 (CaspGLOW™ Red Active Caspase-8 Staining Kit, BioVision) or cleaved caspase-9 (CaspGLOW™ Fluorescein Active Caspase-9 Staining Kit, BioVision) antibodies for 30 min in dark at 37 °C incubator. Expression pattern of above molecules was detected and quantified by NovoCyte flow cytometry and NovoExpress^® software (Agilent Technologies Inc., Santa Clara, CA, USA), respectively. All experiments were performed in triplicate and were repeated at least three times.

FLICA have been designed as affinity labels of the enzyme active centre of caspases44 (link). Use of FLICA allows for a convenient estimation of apoptosis by both cytometry and fluorescence microscopy45 (link). Caspase activity was assessed using a CaspTag Pan Caspase In Situ Assay Kit (Chemicon, Temecula, CA, USA), APOPCYTO Intracellular Caspase-3 or -8 Activity Detection Kit (MBL), and CaspGLOW Fluorescein Active Caspase-9 Staining Kit (BioVision, Milpitas, CA, USA), according to the manufacturers’ instructions. Briefly, 2 × 10⁵ cells were cultured for 60 min in the presence of the substrate, washed, and analysed by flow cytometry.