Calcium colorimetric assay kit

Mouse urine samples were collected at different time points and stored at −20°C until analysis. Urine oxalate concentration was assessed with a colorimetric, enzymatic assay (Oxalate assay kit, Libios) in 96‐well plate according to the manufacturer's instructions. Urine calcium concentration was assessed using calcium colorimetric assay kit (Sigma‐Aldrich), and urinary glucose levels were determined using Glucose GOD FS kit (DiaSys).

Tissues were placed in an oven at 50 °C for 72 h and then reduced to a powder using a glass mortar. Calcium was extracted from 10 to 50 mg of tissue powder (depending on the organs) in 200 µL to 1 mL of a 10% formic acid solution for at least 2 h. The supernatant was recovered after centrifugation at 3000× g for 10 min and then evaporated at 110 °C. Residues were resuspended with mili-Q water corresponding to half of the volume of the formic acid solution. Calcium was assayed using the calcium colorimetric assay kit (Sigma-Aldrich, l’Isle d’Abeau, Chesnes, France), according to the manufacturer’s recommendations.

The calcium ion concentration in H-hPL and Fd-hPL was identified using a calcium colorimetric assay kit (Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer’s protocol. Briefly, H-hPL and Fd-hPL were diluted with MilliQ water to 10× and 100×, respectively, and added into a 96-well-flat bottom plate. The chromogenic reagent was added into standard and sample wells, followed by adding the calcium assay buffer. The mixture was mixed gently and incubated in the dark at room temperature for 5–10 min. The absorbance was read at 575 nm using a spectrophotometric multi-well plate reader (Biotek^®, Santa Clara, CA, USA) with Gen 5 software, version 2.09.

HPLC grade acetonitrile and analytical grade organic solvents, as well as ammonium acetate for sample preparation and HSCCC separation, were obtained from (Sinopharm Chemical Reagent Co. Ltd., Shanghai, China). Ultrapure water was achieved by RU-B water system (Shanghai Tauto Biotech Co. Ltd., Shanghai, China) and filtered through 0.45 μm before use.
Flos Chrysanthemi Indici was bought from the medicinal material market (Haozhou, Anhui, China). Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies (Minato-ku, Tokyo, Japan). Alizarin red-S and calcium colorimetric assay kit were procured from Sigma-Aldrich (St. Louis, MO, USA). The primary antibodies targeting phospho-AKT (Ser473; #4060) and AKT (#4691) were procured from Cell Signaling Technology (Danvers, MA, USA) and Runx2 antibody from Abcam (ab76956; Cambridge, UK).

About 10,000 cells/well HOBs were seeded overnight in a 96-well plate in the treatment medium. Then, the cells were induced with a treatment medium containing (i) media only as the negative control; (ii) 100 µM inorganic pyrophosphate (positive control); and (iii) 10, 25, 50, and 100 µg/mL protein oxHDL in 100 µL of media (final total volume) for 14 days. The media was changed every 4 days. Then, on the 14th day of incubation, the cells were washed twice with PBS (phosphate-buffered saline). About 0.6 N HCL (100 µL) was added to each well to decalcify the cells. After 24 h, the supernatants were collected to quantify the calcium content using a Calcium Colorimetric Assay Kit (Sigma-Aldrich, St. Louis, MO, USA) while the protein content inside the cells was collected by solubilizing the cells with 0.1 N NaOH. The amount of calcium was normalized with the cells’ total protein content and quantified using a BCA protein assay kit (Thermo Fisher Scientific, Pierce, IL, USA).

For determination of total calcium content, samples (n = 3) were washed twice with PBS (Gibco) and extracted off a well of a 96-well plate in 0.5 M HCl solution (Sigma-Aldrich). Accumulated calcium was removed from the cellular component by shaking overnight at 4 °C. The consequent supernatant was utilized for calcium determination according to the manufacturer’s instructions contained in the calcium colorimetric assay kit (Sigma-Aldrich). Total calcium was calculated from calcium standard solution prepared in parallel. Absorbance at 575 nm was measured for each condition and normalized to the total number of cells, after 21 days of osteogenic differentiation.

Antibodies for Arg1 (Cat#: ab212522), CD31 (Cat#: ab28364) and ABCG1 (Cat#: ab52617) were purchased from Abcam (Cambridge, MA). Antibodies for IL-1β (Cat#: sc-52012), ICAM-1 (Cat#: sc-107), VCAM-1 (Cat#: sc-13160), ALP (Cat#: sc-365765), Οsx (Cat#: sc-393325) and RUNX2 (Cat#: sc-390351) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies for Cleaved Caspase1 (Cat#: 4199), ABCA1 (Cat#: 96292), β-actin (Cat#:4970) were purchased from Cell Signaling Technology (Danvers, MA). TG assay kit (Cat#:100020090), total cholesterol assay kit (Cat#: 100020080), LDL-C assay kit (Cat#:100020245), HDL-C assay kit (Cat#:100020235) were purchased from Biosino Bio-Technology and Science INC (Beijing, China). Alizarin Red S solution (Cat#: G3280) was purchased from Solarbio & Technology Co., Ltd (Beijing, China). IL-1β Elisa kit (Cat#: SEA563Mu), IL-6 Elisa kit (Cat#: SEA079Mu), TNF-α Elisa kit (Cat#: SEA133Mu), IL-10 Elisa kit (Cat#: SEA056Mu) and SOD Elisa kit (Cat#: SES134Mu) were purchased from Cloud-Clone Corp. (Wuhan, China). Mouse ROS Elisa kit (Cat#: YX-181519M) and Mouse ox-LDL Elisa kit (Cat#: YX-152412M) were purchased from Sino Best Biological Technology CO., Ltd (Shanghai, China). Calcium Colorimetric Assay kit (Cat#: MAK022-1KT) was purchased from Sigma-Aldrich. All other reagents were purchased from Sigma-Aldrich except where indicated.