Calcium detection assay kit

LVPM cells were cultured in osteogenic medium containing DMEM/Low Glucose (Invitrogen, Carlsbad, CA) with 0.1 μM dexamethasone and 50 μM ascorbic acid 2-phosphate. On day 9, β-glycerolphosphate (10 mM) was added to induce mineralization. On day 11, calcium content was evaluated by a Calcium Detection Assay kit (Abcam, Cambridge, UK) according to the company manual. On day 21, Alizarin Red staining was performed in order to identify accumulation of mineralized calcium. The wells were washed twice with PBS, and briefly, cells were fixed with 70% ethanol (Sigma-Aldrich, St. Louis, MO) for 30 min. After fixation, the wells were stained with 0.2% Alizarin Red S solution (pH 4.2; Sigma-Aldrich, St. Louis, MO) for 30 min. For the final wash, each well was washed with PBS (Gibco Invitrogen, Grand Island, NY) three times [15 ].

The calcium concentration was measured using a Calcium Detection Assay Kit (Abcam) according to the manufacturer's protocol. Liver tissues (100 mg) were homogenized in 500 µl of calcium assay buffer and then sonicated for 10 s in an ice bath with a VCX‐500 Ultrasonic Processor (Sonics and Materials Inc.). The homogenate was centrifuged at 12000 × g for 5 min (Eppendorf Centrifuge 5424R, Eppendorf AG). After centrifugation, the supernatant was transferred to a clean tube and used as the tissue lysate. Calcium standards were prepared in serial dilutions of 0, 0.2, 0.4, 0.6, 0.8, and 1 mM by diluting the 5 mM standard stock in distilled water. The reaction was set up by mixing 50 µl of calcium standard or tissue lysates, 90 µl of chromogenic reagent, and 60 µl of calcium assay buffer into a 96‐well plate, and then incubating for 10 min at room temperature in the dark. The absorbance at 575 nm was measured using a VERSAmax™ microplate reader (Molecular Devices). The calcium concentration (mM) was calculated by dividing the sample amount from the standard curve by the sample volume added into the well and multiplying by the sample dilution factor.

For evaluation of intracellular Ca²⁺ level, cells were cultured in six-well plates at 80% confluence and grown under the indicated condition. Then, cells were harvested and suspended in 500 μl calcium assay buffer and put on ice for quickly pipetting a few times, and subsequently centrifuged at 15000 g at 4 °C for 10 min to obtain supernatants. Ca²⁺ level in the supernatants was evaluated using the Calcium detection assay kit (Abcam; catalog# ab102505) according to the manufacturer’s instruction. The experiment was repeated five times.

The concentration of Ca²⁺ was measured using the Calcium Detection Assay Kit (Abcam). The samples were pre-treated with the ESPs of A. cantonensis L5 and then treated with Chromogenic Reagent and Calcium Assay Buffer at room temperature for 10 min protected from light. After incubation, the samples and standards were analysed with a spectrophotometer (OD575 nm).

Levels of calcium in the cytoplasm of BV2 cells were measured using calcium detection assay kit (Abcam). HT22 neuronal cells were seeded into 24-well at a density of 2 × 10⁵ cells/ml and allowed to settle. At confluence, the medium was replaced with conditioned medium as explained previously and incubated for 24 h at 37 °C. Later, lysates were collected using RIPA lysis buffer followed by centrifugation at 4 °C, 13,500 rpm for 15 min. Fifty microlitres of the lysates was pipetted into 96-well plate followed by 90 μl of chromogenic reagent along with 60 μl of calcium assay buffer, incubated in the dark for 15 min at room temperature. Output was measured using Tecan F50 microplate reader at a wavelength of 575 nm. Concentrations of calcium in the cytoplasmic lysates were calculated by comparing to the standard curve (0–2.0 μg/ml).