Spectramax 384 microplate reader

For LUV preparation, 100 µl of a MIM-mimic lipid mixture (egg PC/egg PE/liver PI/brain PS/heart CL = 40:34:5:3:18 [weight ratio] dissolved in 4:1 chloroform/methanol [10 mg/ml]) was dried under a stream of nitrogen and rehydrated with 500 µl of 50 mM CF, 100 mM sucrose, and 5 mM Hepes/KOH, pH 8. Multilamellar liposomal suspensions were extruded through a 0.1-µm polycarbonate membrane using a Mini Extruder (Avanti Polar Lipids) and purified on a PD-10 desalting column (GE Healthcare). The standard external buffer was 150 mM KCl and 10 mM Hepes/Tris, pH 7. The osmotic pressures of all experimental buffers were adjusted to ∼300 mOsm with sucrose using a VAPRO osmometer (Wescor Inc.). For CF-LUVs incubated with 10 mM CaCl₂ or MgCl₂, 135 mM KCl were used to adjust the osmotic pressure. LUVs treated with 90% trifluoroethanol (TFE) were used as a control. The final TFE concentration was restricted to 0.9%, a percentage at which no significant CF release was observed. CF leakage was analyzed at λ 488-nm excitation and 517-nm emission using a SpectraMax 384 microplate reader (Molecular Devices) and calculated using the formula CF leakage (%) = 100 × (F − F₀)/(F_max − F₀), where F is the measured fluorescence intensity, F₀ is the fluorescence intensity of intact LUVs, and F_max is the fluorescence intensity of LUVs treated with 0.1% Triton X-100.

For the CCK-8 assay, ADSCs were inoculated at 5x10⁴/well in 24-well plates coated with or without d-ECM in 500 μL medium containing 0% or 2% foetal bovine serum (FBS, GUXMX-90,031, Cyagen Biosciences, USA), and then cultured for different time (1, 2, 3, 4 days). The cells growing on the plastic substrates in 10% FBS were used as control group. After the indicated culture time, cells in each well were washed with PBS twice and then incubated in 500 μL serum-free medium containing 10% CCK-8 regent (CK04, Dojindo Molecular Technologies, JPN) for 4 h at 37°C. Sequentially, the plates were sufficiently shaken, and a total of 400 μL mixture for each sample was equally transferred into 4 wells of a 96-well plate. Absorbance values at 450 nm were measured using a SPECTRA max 384 microplate reader (Molecular Devices Corporation, USA) and were then normalized by control group.
For cell counting, cells were seeded at 5x10⁴/well on the plastic or d-ECM substrates of 24-well plates and then cultured in 2% or 10% FBS for 3 days. Afterwards, cells were collected and counted using a Countess II Automated Cell Counter (Thermo Fisher Scientific, USA).

Mouse primary chondrocytes were cultured as was described above. Primary chondrocytes of each group were moved to tissue-culture flasks with DMEM (containing 25mM HEPES, 2mM glutamine, 100μg/ml streptomycin, 100IU/ml penicillin, 2.5μg/ml gentamicin and without serum), with or without the treatment of 10 ng/ml TNF-α and 50 μg/ml CST. After incubating for 24 h, the conditioned medium was collected for GAG synthesis analysis. Conditioned medium was collected from each experimental group, and the presence of GAG released from primary chondrocytes was quantified using dye DMMB (Polysciences, Warrington, PA, USA). Culture medium was pretreated with 0.5 units/ml of hyaluronidase (Seikagaku, Tokyo, Japan) at 37 °C for 3 h in order to get rid of exogenous HA in order to remove interference. Digests were mixed with DMMB in 96-well plates and read at 520 nm with Spectra Max 384 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). GAG amount in the conditioned medium was extrapolated with standard chondroitin-6-sulfate sodium salt (Sigma e Aldrich, St. Louis, MO, USA).