Calcein am pi assay kit

Gelatin (300 KDa, Type A, from porcine skin) was purchased from VETEC. Polyethylene glycol (PEG) (2000 KDa), 4-(dimethylamino) pyridine (DMAP), methacrylic anhydrideand and Irgacure 2959 were obtained from Sigma-Aldrich (St. Louis., MO, USA). 4-formylbenzoic acid (AR, 98%), N, Nʹ-dicyclohexylcarbodiimide (DCC) and 2, 4, 6-trinitrobenzenesulfonic acid (TNBS) were purchased from Aladdin Reagent Company (Shanghai, China). Multi-walled carbon nanotubes (MW-CNTs) were purchased from Chengdu Organic Chemicals Co. Ltd., Chinese Academy of Sciences. Dulbecco’s Modified Eagle’s Medium (DMEM), CCK-8 Cell Proliferation and Cytotoxicity Assay Kit, and Calcein-AM/PI Assay Kit were acquired from Beijing Solarbio Science & Technology Ltd (China). MC3T3-E1 Cell was purchased from American Type Culture Collection (Manassas, VA). Tetrahydrofuran (THF) was distilled prior to use, and other reagents were of analytical grade and without further purification.

Live and dead cell staining was performed using a Calcein-AM/PI assay kit (Solarbio, China). Treated cells were stained with calcein-AM solution (1:1000) and incubated at 37℃ for 20 min, then added PI solution (3:1000) incubated at 37℃ for 5 min. The numbers of live and dead cells were detected under a laser confocal scanning microscope (OLYMPUS IX83, USA). The live cells fluoresced green (Calcein-AM staining), and the dead cells fluoresced red (PI staining). Under 400X magnification, 6 fields of view were taken for each hole.