Quantichrom calcium kit

Chondrocytes were cultivated for 24 h in DMEM with 10%FBS, supplemented with Secondary calciprotein particles (CPP) (50 μg/ml calcium) to induce calcification (Aghagolzadeh et al., 2017 (link); Nasi et al., 2020 ). Cells were fixed in 10% formol and calcium-containing crystals stained with Alizarin red (Gregory et al., 2004 (link)). Calcium content was quantified by the QuantiChrom^TM Calcium Kit (BioAssay Systems) by reading absorbance at 612 nm using the Spectramax M5e reader (Molecular Devices).

Extracellular vesicles of 30–150 nm diameter (corresponding to matrix vesicles, MVs) were isolated from chondrocytes supernatants by serial ultracentrifugations at 4 °C [8 (link)] using Optima™ XPN-80 (Beckman Coulter, Brea, CA, USA). Briefly, WT and CD11b KO chondrocytes were cultured for 24 h in DMEM with 10% FBS. Supernatants were collected and centrifuged at 300× g for 10 min to remove dead cells and debris. Then, a second centrifugation at 2000× g for 10 min was performed to pellet apoptotic bodies (1000–5000 nm). A third centrifugation at 10,000× g for 30 min allowed us to pellet and remove larger microvesicles (>100–1000 nm). The final centrifugation at 100,000× g for 70 min was done to pellet matrix vesicles. Pelleted MVs were resuspended in PBS, and RIPA buffer was added to disrupt the phospholipidic layer in order to measure calcium content with QuantiChromTM Calcium Kit (BioAssay Systems, Hayward, CA, USA).