Cell counting kit 8

Mouse pre-osteoblasts (MC3T3-E1) cells were provided by Peking Union Medical College Hospital. MC3T3-E1 grows in a culture medium containing 10% (volume) fetal bovine serum in DMEM (GE Life Science-Hyclone) medium with 100 U/ml penicillin and 100 mg/l streptomycins. The culture medium was stored in a cell incubator with 37 °C and 5% CO₂ and changed every 2 d. The samples of TZNF, TZNF-b1 and TZNF-b2 were soaked in ethanol for 2 h and placed under ultraviolet light for light sterilization. Then, the three samples were put in a 24-well plate to co-culture with MC3T3-E1 for 7 d and inject 1 mL of 1 × 10⁴ cell/mL cell suspension into each well. The illuminated samples were taken out and exposed to light for 0.5 h under the NIR lamp every day. The proliferation of the cells co-cultured with the sample was tested on the 1, 3 and 7 d using Cell Counting Kit (CCK)-8 (Beyotime, China). The optical density of the solution at 450 nm was measured by the microplate reader. The samples cultured for 3 d were fixed with 2.5% glutaraldehyde, and then stained with Actin-Tracker-Red and DAPI for 30 and 15 min, then placed under a fluorescence microscope to observe and take photos.

Tetraethyl orthosilicate (TEOS), triethyl phosphate (TEP), calcium nitrate trihydrate (Ca(NO₃)₂•3H₂O), Cerium nitrate hexahydrate (Ce(NO₃)₃•6H₂O), Dodecylamine (CH₃(CH2)₁₁NH₂) were purchased from Aladdin Industrial Corporation. Gelatin from porcine skin, Methacrylic anhydride (MA) and Lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate (LAP) were purchased by Sigma (Sigma-Aldrich, St. Louis, Missouri, US). Cell counting kit (CCK-8) was purchased from Beyotime Biotechnology Co. Ltd., (Beijing, China). The Live & Dead^TM Viability/Cytotoxicity Assay Kit for Animal Cells (Calcein AM, EthD-1) was purchased by US EverBright, (Jiangsu, China). Hematoxylin and eosin (H&E) was purchased by Shandong Sparkjade Biotechnology Co. Ltd., (Shandong, China). Masson’s Trichrome was purchased by Solarbio Science&technology Co. Ltd., (Beijing, China).

After transfection, SW1990 and Panc-1 cells were seeded at 4 × 10³ cells per well in a 96-well plate and incubated for 24 h, 48 h, 72 h, and 96 h, and then, 10 μL of Cell Counting Kit- (CCK-) 8 (Beyotime, China) was added to each well. The plates were then incubated at 37°C for 2 hours. A microplate reader (Tecan, Mannedorf, Switzerland) was used to measure the OD value of each well at 450 nm. For colony formation assay, SW1990 and Panc-1 cells were seeded into 6-well plates. After transfection, cells were cultured for 2 weeks until most colonies were visible to the naked eye. Then, SW1990 and Panc-1 cells were fixed with 4% paraformaldehyde for 20 minutes and stained with 0.1% crystal violet to observe and count the number of colonies.