Alkaline phosphatase assay kit

Tension group and no-tension group cells were washed by ice-cold PBS twice and fixed in 1 ml 4% paraformaldehyde for 20 min at room temperature. Alkaline phosphatase (ALP) staining was performed using BCIP/NBT Alkaline Phosphatase Color Development Kit (C3206; Beyotime, Shanghai, China) according to the manufacturer’s protocol. Then, photos were taken by a scanner (GE Image scanner III). Cellular ALP activity was detected using Alkaline Phosphatase Assay Kit (A509-2; Jian-Cheng Bioengineering Institute, Nanjing, China) and the procedure was described before (Li et al., 2020 (link)). Finally, the absorbance values related to ALP activity were recorded at 520 nm with the microtiter plate spectrophotometer (Spectrama).

Tetraethyl orthosilicate (TEOS), branched polyethyleneimine (PEI, 25 kDa), N-hydroxysuccinimide (NHS), cetyltrimethylammonium chloride solution (CTAC), triethylamine (TEA), and dimethyl sulfoxide (DMSO) were purchased from Sinopharm Chemical Reagent (Shanghai, China). VEGF was obtained from PeproTech (Cranbury, NJ, USA). DMEM, trypsin, type Ⅱ collagenase, penicillin/streptomycin, fetal bovine serum, and Alizarin red were purchased from Sangon Biotech (Shanghai, China). Porcine aortic valves were purchased from Songlin Meat Food Co., Ltd. A Cell Counting Kit-8 (CCK-8) and a BCIP/NBT ALP staining kit (Beyotime, Shanghai, China). An alkaline phosphatase assay kit (Jiancheng Bioengineering Institute, Nanjing, China), a calcium content detection kit (MingDian, Shanghai, China), and an RNeasy mini kit were purchased from Qiagen (Duesseldorf, Germany), IL-1, IL-6, IL-10, and TNF-α kit (R&D, Santa Clara, CA, USA). Primary antibodies (GAPDH, OPN, OSX, RunX2, α-SAM, Vimentin, and CD31) and donkey anti-rabbit IgG were procured from Abcam (Cambridge, UK). LipofectamineTM 3000 Transfection Reagent was purchased from Invitrogen (Carlsbad, CA, USA).