Victor 1420multilabel counter microplate reader

Cell viability was analyzed with Live/Dead fluorescence staining (Thermo Fisher Scientific) at 3 and 14 days, as described previously [25] . The living cells were stained with 0.5 mM calcein acetoxymethyl ester (green stain) and necrotic cells were stained with 0.25 mM ethidium homodimer-1 (red stain) for 45 min in RT. The samples were imaged using an epifluorescence Olympus IX51 microscope and Olympus DP30BW digital camera (Olympus, Tokyo, Japan).
Cell number was measured based on the total amount of DNA with CyQUANT Cell Proliferation Assay Kit (Thermo Fisher Scientific) according to the manufacturer's protocol at 14 and 21 days.
Briefly, the cells were lysed with 0.1 % Triton X-100 buffer (Sigma-Aldrich) and the hydrogel samples were homogenized mechanically by the Ultra-Turrax tissue homogenizer (IKA Labortechnik, Staufen, Germany). The lysed samples were stored at -80°C until analysis after a freeze-thaw cycle. A working solution was prepared with the kit provided CyQUANT GR dye and Cell lysis buffer. Fluorescence of 3 parallel samples was measured at 480/520 nm with Victor 1420
Multilabel Counter microplate reader (PerkinElmer, Waltham, MA, USA).

For the hASC mineralization assay, the hASC secreted hydroxyapatite residues of the cell matrix in 3D hydrogel were stained with the OsteoImage assay according to manufacturer's protocol (OsteoImage Mineralization Assay; Lonza) at 21 days [26] (link). Briefly, the cells were fixed with 4 % PFA (Sigma-Aldrich) for 30 min in RT and the hydroxyapatite residues were stained with the OsteoImage Staining Reagent for 45 min and immediately measured at 490/535 nm with Victor 1420 Multilabel Counter microplate reader (PerkinElmer). After quantitative measurement, the cell nuclei were stained with DAPI (dilution 1:2000) and detected at 361 nm together with the stained hydroxyapatite residues imaged at 492/520 nm with an epifluorescence Olympus IX51 microscope and Olympus DP30BW digital camera (Olympus).