Spectramax m5e reader

Chondrocytes were treated with CPP (50 μg/ml calcium) for 24 h or calcifying medium for 24 h, then washed in PBS and fixed in 10% formaldehyde. Crystals were quantified with Alizarin Red staining with Adobe^® Photoshop^® after image binarization and related pixel counts. Calcium content was quantified by the QuantiChromTM Calcium Kit (BioAssay Systems) by reading absorbance at 612 nm using the Spectramax M5e reader (Molecular Devices).

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).

Fluorescence polarization assays were performed manually in black 96 microplate as previously described (Miyoshi et al. 2016 (link)). Each well (100 μL) contained 5 nM 3′-6-FAM-labelled oligonucleotides and varied concentrations (0–1.5 μM) of PsPIWI-RE in reaction buffer (20 mM Tris–HCl (pH 7.5), 250 mM NaCl, and 5 mM MgCl₂) for 30 min at 37 °C. The polarisation values were measured with excitation at 494 nm, emission at 522 nm in a SpectraMax M5e reader (Molecular Devices, CA, USA). The data were fitted to a nonlinear regression curve using the one-site-specific binding function in GraphPad Prism 8. All experiments were performed in triplicate.