Cyquant cell proliferation assay

CyQuant cell proliferation assay (Invitrogen/Life Technologies Corp, Carlsbad, CA) was performed as previously reported [11 (link)] and following the manufacturer’s instructions. Relative number of viable cells was normalized to the DMSO-treated control cells.

An enzyme-linked immunosorbent assay (ELISA) was used to study the factors secreted by the human macrophages when cultured on the implant surfaces. Commercially available ELISA DuoSet Development Kits (R&D Systems, McKinley, Minneapolis, United States) were used to measure the secretion levels of the pro-inflammatory cytokine IL-6 and tissue-repair related chemokine CCL18 present in the supernatant. Therefore, the collected medium at day 3 for 3 different donors was centrifuged for 5 min at 500 g and were stored at −80°C until the assay was performed according to the manufacturer’s instructions. An ELISA assay was also performed for the macrophages with co-cultured the hMSCs: after 3 days of indirect co-culture, the macrophages were transferred to a non-adherent 24 well plate with 500 µL of the co-culturing medium. After 24 h, 400 µL of the cell supernatant was collected and stored at −80°C. The protein secretion levels of the (co-cultured) macrophages were normalized to the DNA content of the macrophages attached to each implant. Therefore, the implants with adhered macrophages were harvested at the same time with the medium collected for ELISA and were stored at −80°C. The DNA quantification was performed using a CYQUANT cell proliferation assay (Invitrogen, Carlsbad, California, United States) following the manufacturer’s instructions.

We used CyQUANT Cell Proliferation Assay (Invitrogen), which can accurately quantify the entire cell population with DNA specific dye that exhibits strong fluorescence enhancement when bound to DNA, following the manufacturer’s protocol. Briefly, cells were plated in a 96-well plate at a density of 5000 cells per well and incubated overnight at 37 °C. The next day, the cells were transfected either with scramble control or miR-124 mimic as described above. Then, the cells were exposed to As for 48 h. After removing medium from wells, plates were frozen at −70 °C. The plates were thawed at room temperature, and then we added 200 μL of the CyQUANT GR dye/cell-lysis buffer to each sample well. After incubation for 2–5 min, the fluorescence was measured using a microplate reader at 480 nm(excitation)/520 nm (emission).

Glucose, lactate, glutamine, and glutamate concentration were measured using Glucose-Glo™ Assay, Lactate-Glo™ Assay and Glutamine/Glutamate-Glo™ Assay (Promega), respectively. Culture medium dilution of 1/500, 1/100 and 1/50 in PBS were used to measure glucose, lactate, and glutamine/glutamate concentration, respectively. Cell ATP concentration was measured using CellTiter-Glo® Luminescent Cell Viability Assay (Promega), following the manufacturer’s instructions. Cell DNA content was measured using CyQUANT™ Cell Proliferation Assay (Invitrogen) by resuspending cell pellets in 250 μL CyQUANTTM GR dye/cell-lysis buffer. Finally, cell protein content was measured by resuspending cell pellets in PathScan® Sandwich ELISA Lysis Buffer (Cell Signalling Technology) then by using Pierce™ 660 nm Protein Assay Reagent (Thermo Scientific™) following the manufacturer’s instructions. For all these assay, absorbance, fluorescence, and luminescence were read with SpectraMax® M3 plate reader (Molecular Devices).

Five micropellets of BM-MSCs transfected with siCT or siNMB were recovered at day 21 of chondrogenesis and digested in 300 µL of papain (592 µg/mL) in a solution of sodium acetate buffer (0.1 M, pH 5.5), EDTA (0.5 M), L-cystein-HCl (5 mM)) at 60 °C during 18 h. Forty microliters were used for GAG quantification, 90 µL for collagen quantification and 60 µL for DNA quantification. GAG, total collagens and DNA were measured using the Glycosaminoglycan Assay Blyscan™ (Biocolor life science assays, Interchim), the Soluble Collagen Assay Sircol™ (Biocolor life science assays, Interchim) and the CyQUANT™ Cell Proliferation Assay (Invitrogen), respectively, following manufacturer’s instructions.