Hydrochloric acid (hcl)

Materials including 2,6-dimethyl-2,6-octadiene aldehyde (citral, 97%), tetraethyl orthosilicate (TEOS, 98%), cetyltrimethylammonium bromide (CTAB, 99%), aqueous ammonia (28%), 3-aminopropyltriethoxysilane (APTES, 97%), absolute ethanol (99.8%), hydrochloric acid (10%), 2,4-dinitrophenylhydrazine test solution (98%), trypsin soybean broth (TSB), and phosphate buffer saline (PBS, 0.1 M, pH = 7.4) were all provided by Sangon Biotech (Shanghai, China).

The strain of silkworm we used in this work was a commercial silkworm strain 872. Silkworms were reared in our laboratory with fresh mulberry leaves at 25 °C and 75% relative humidity under 12 h photoperiod. Fresh silkworm cocoons were collected for sericin preparation. Dopamine hydrochloride and silver nitrate (AgNO₃) were purchased from Aladdin (Shanghai, China). Hydrochloric acid and Tris (hydroxymethyl) aminomethane (Tris) were from Sangon Biotech (Shanghai, China). Ultrapure water made by a MilliQ water purification system (Millipore, Billerica, MA, USA) was used in the experiment. The Cell counting kit-8 (CCK-8) used in the experiment was bought from Beyotime (Beijing, China). LIVE/DEAD cell viability kit was bought from Thermo Fisher Scientific (Waltham, MA, USA). NIH3T3 (mouse embryonic fibroblast) cell lines were received from the China Infrastructure of Cell Line Resources. The Dulbecco’s modified Eagle’s medium (DMEM), Penicillin/Streptomycin, Fetal Bovine Serum (FBS), and Trypsin-EDTA were bought from Gibco BRL (Gaithersburg, MD, USA).

The samples were intervened with drug and the culture medium was removed. Followed by three cycles of PBS washing, 2 mL, 0.6 mmol/L of hydrochloric acid (HCL) (Sangon Biotech, China) was supplied to each hole of 6-well plates for incubation 24 h at room temperature, collected the supernatant, and determined calcium concentration at 24 h. The calcium concentration was detected by QuantiChrom^TM Calcium Assay Kit according to the manufacturers’ instructions (QuantiChrom, USA). Of 5 μL sample was added to 96-well plates, supplemented with 200 μL of working solution, and vibrated at room temperature for 3 min. The absorbance at 612 nm was measured using a CMax Plus microplate reader (Thermo, USA). Based on the obtained concentration, we calculated the total amount of calcium ions in the supernatant of all groups. The formula used for calculation was presented: Calcium ions (μg) = Calcium ion concentration in the supernatant (mmol/L) × 40 × 10³ μg/L × 2 × 10⁻³ L. Total protein contents of each group were also calculated using the BCA method. Calcium ion contents per milligram of protein in each group was obtained when the total amount of calcium ions in each group was divided by total protein content.