3 4 5 dimethyl 2yl 2 5 diphynyltetrasolium bromide mtt

Dulbecco’s modified Eagle’s medium (DMEM) and penicillin/streptomycin solution were purchased from GIBCO (Grand Island, NY, USA). Fetal bovine serum (FBS) was obtained from Merck (Sacramento, CA, USA); dimethyl sulfoxide (DMSO) and 3-(4-5-dimethyl-2yl)-2-5-diphynyltetrasolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The intracellular NO production was detected using DAF-FM DA (Thermo Fisher Scientific, Waltham, MA, USA); the total NO production was measured using the Griess assay (Promega Corporation, Madison, WI, USA). Calcium levels were quantified using Fluo-4-AM dye (1-[2-amino-5-(2,7-difluoro-6-hydroxy-3-oxo-9-xanthenyl) phenoxyl]-2-(2-amino-5-methylphenoxy) ethane-N, N, N′, N′-tetraacetic acid, pentaacetoxymethyl ester) (Thermo Fisher Scientific, Waltham, MA, USA ). Atropine, a specific AchR antagonist, was purchased from Sigma Aldrich. SU5416, a VEGFR2 inhibitor, was obtained from Tocris Bioscience (Bristol, UK).

Alpha Minimum Essential Medium Eagle (α-MEM), penicillin-streptomycin, and fetal bovine serum (FBS) were purchased from Gibco BRL (Burlington, ON, Canada). Dimethyl sulfoxide and 3-(4-5-dimethyl-2yl)-2-5-diphynyltetrasolium bromide (MTT) were obtained from Sigma-Aldrich (St, Louis, Mo, USA). Trizol reagent was purchased from the molecular research center (Montgomery, OH, USA). cDNA synthesis kit was purchased from Promega Co. (Madison, WI, USA). Other chemicals and regents used were of the highest grade available commercially. The SHE used in this study was equal to the sample prepared from S. horneri in the previous study [11 (link),12 (link)].

The ERM-certified PM material (ERM-CZ100 and PM₁₀-like); dimethyl sulfoxide (DMSO); azo dye-impregnated collagen; fluorescent probe 2′; 7′-dichlorodihydroflurescin diacetate (DCFH-DA); 3-(4-5-dimethyl-2yl)-2-5-diphynyltetrasolium bromide (MTT); and MMP-1, 2, 8, 9, and 13 ELISA kits were purchased from Sigma-Aldrich (St. Louis, MO, USA). The phosphate-buffered saline (PBS, 1X), Dulbecco’s modified Eagle medium (DMEM), Ham’s Nutrient Mixtures medium (F-12), fetal bovine serum (FBS), and penicillin/streptomycin (P/S) were purchased from Gibco BRL (Life Technologies, Burlington, ON, Canada). Antibodies against NF-κB p50 and p65; phosphorylated AP-1 (p-c-Jun); nucleolin; MAPKs including p-ERK, p-JNK, and p-p38; and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-mouse and anti-rabbit IgG antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). PIP EIA kit was purchased from TaKaRa Bio Inc. (Shiga, Japan). All other chemicals used in this study were analytical grade.