Multiscan ex reader

The antioxidant activity of test samples was measured by using our established microtiter-based DPPH assay [15 (link)]. Briefly, a DPPH solution (0.1 mM, in methanol) was incubated with varying concentrations of test compounds for 20 min at room temperature, and the absorbance of the resulting solution was read at 550 nm against a blank using a Multiscan EX Reader (Thermo Labsystems, Altrincham, UK).

The Sulforhodamine B (SRB) assay was performed to determine whether the DEHP treatment affected the number of hCGC, as previously described [27 (link)]. Briefly, the cells were plated in a 96-well plate, treated for 48 h with DEHP, and subsequently fixed for 1 h at 4 °C by adding 50 μL of 50% (w/v) trichloroacetic acid per well. After fixation, the cells were washed five times with distilled water and stained with 50 μL of 0.4% SRB in 1% acetic acid for 30 min. After staining, the cells were washed five times with 1% acetic acid and air-dried. The stain was solubilized in 10 mM Tris (hydroxymethyl) aminomethane (pH 10.5), and the light absorption was measured using a Thermo Labsystems Multiscan EX reader set at 492 nm, with a reference wavelength at 690 nm. The samples for the analysis were run in duplicate.

For osteogenic differentiation assays, 3x10⁴ culture-expanded MSCs (passage 3) from donor-matched IC-BM and VB-BM samples were cultured in osteogenic media. The osteogenic media was formed of low glucose DMEM (ThermoFisher Scientific Waltham, MA, USA) supplemented with 10% FCS (ThermoFisher Scientific), penicillin and streptomycin (Thermo Fisher Scientific), 100nM dexamethasone, 10mM β-glycerophosphate and 0.05mM ascorbic acid (all from Sigma-Aldrich). After 14 days of culture, the quantification of the calcium level was performed using colorimetric calcium kit (Calcium Liquid, Sentinel Diagnostics, Milan, Italy) as previously described [37 (link)]. To extract calcium, cultured MSCs were treated with 1M of HCl solution. The spectrophotometric reading was taken on MULTISCAN EX reader and analysed using Ascent software (Thermo Fisher Scientific). Additionally, the staining for calcium deposition and alkaline phosphatase (ALP) expression was performed using Alizarin Red dye and fast blue RR salt dye respectively (both from Sigma-Aldrich) as used previously [32 (link), 38 (link)]. The culture plates were scanned using an Epson 3590 flatbed scanner (Epson Ltd, Hertfordshire, UK).