Assay buffer

To measure lysyl oxidase (LOX) activity, supernatants were obtained 7 days after the induction of MSCs to OBs. The LOX assay reaction solution is a mixture of 20 μl Amplite™ HRP substrate stock solution (Abcam), 20 μl HRP (50 U/ml, Abcam), and 5 ml assay buffer (Abcam). A total of 50 μl supernatant and 50 μl LOX assay reaction solution were added to 96 wells. After incubation at 37 °C for 20 min in the dark, fluorescence was measured at Ex/Em = 540/590 using a microreader (Tecan, Maennedorf, Switzerland).

Based on the protocol summary of Fe²⁺ assay, the samples were placed into 96-well plates. Each well was added with 5 µL assay buffer (Abcam, Cambridge, MA, USA) and incubated for 30 min at 37°C. Then, 100 µL Iron Probe (BioAssay Systems, CA, USA) was added to each well containing the Iron Standard and test samples and incubated for 1 h at 37°C protected from light. The absorbance at 593 nm was analyzed immediately with a colorimetric microplate reader (Bio-Rad Laboratories, Shanghai, China).

glutamine uptake and α-KG production were determined by glutamine and α-KG assay kits (Abcam, Cambridge, UK), respectively. In brief, cultured cells were harvested and then suspended in assay Buffer (Abcam). After that, samples were centrifuged at 9000 rpm for 12 min, and supernatant was collected. Perchloric acid (Abcam) and potassium hydroxide (Abcam) were used to incubate with supernatant, respectively. After performing centrifugation at 12,000 rpm for 12 min, supernatant was collected and analyzed with the microplate reader (Thermo Fisher) with the wavelength at 450 nm for glutamine uptake assay or at 570 nm for α-KG production assay.

The cellular tyrosinase activity were measured using tyrosinase Activity Assay Kit (Abcam, Cambridge, UK) and a slight modification of a previously reported method [40 (link)]. B16-F10 cells were seeded in 60 mm dishes (2 × 10⁵ cells/dish) for 24 h and treated with CFS (0.5 and 1% (v/v)) or arbutin (200 µM) for 40 h in the presence or absence of 200 nM α-MSH, harvested by trypsinization, sonicated in assay buffer (Abcam, Cambridge, UK), and centrifuged at 12,000 rpm for 20 min. The protein concentration was determined by the Pierce™ BCA Protein Assay Kit (Thermo Fischer Scientific). The reaction mixture consisting of 20 µg protein and 80 µL of 2 mg/mL L-DOPA (in 0.1 M sodium phosphate buffer, pH 6.8) was added to each well of a 96-well plate. After incubation at 37 °C for 1 h, the optical density at 492 nm was measured using a microplate reader.

Glutamate production was detected with glutamate assay kit (Abcam). Briefly, cells were collected when their confluence reached about 80%. Then, harvested cells were incubated with lysis buffer (Abcam) for 15 min. After that, Assay buffer (Abcam), Enzyme mixture (Abcam) and NADP Stock Solution (Abcam) were mixed with lysates. Finally, samples were analyzed with the microplate reader (Thermo Fisher) with the wavelength at 570 nm.

Culture supernatants were collected 2 to 3 times a week and kept at − 80 °C until the assays were performed. All experiments were performed according to the manufacturers’ protocols. In brief, released IL-1β, IL-6, and TNFα were measured from undiluted supernatants using commercially available V-Plex rat ELISA kit (Meso Scale Discovery, Rockville, MD), and electroluminescent signals were detected by MESO QuickPlex (Meso Scale Discovery, Rockville, MD). Lower detection limit of the kit was 6.92 pg/ml for IL-1β, 13.8 pg/ml for IL-6, and 0.72 pg/ml for TNFα. Lactate dehydrogenase (LDH) was measured using LDH assay kit (BioVision, Milpitas, CA). The culture supernatants were diluted two- to threefolds with the assay buffer (BioVision), and colorimetric signals were measured at 450 nm by a microplate reader FlexStation 3 (Molecular Devices, San Jose, CA). LDH concentrations were calculated from the level of LDH activity that converts NAD to NADH. The concentration was expressed as milliunits per milliliter, in which 1 U of LDH generates 1 μM NADH per minute. Goat immunoglobulin G concentration in culture supernatants was measured using the competitive inhibition IgG ELISA kit (Cusabio, College Park, MD). The detection range of the kit was 0.146 to 37.5 μg/ml.