Celltiter 96 aqueous cell proliferation assay

4 types of mGL/mHA monomer solution were prepared as described above. P3 hBMSCs pellets were re-suspend using monomer solution with a final density of 20×10⁶/ml. The suspension was then poured into a mold (cylindrical void with 2mm height and 5mm diameter) and subjected to 4-minute visible light exposure, using a dental curing lamp. The cell-laden gelatin hydrogels were extracted and cultured in chondrogenesis-inducing medium (DMEM with high glucose (Gibco, 11995), 1% Antibiotic-Antimycotic, 0.1 μM dexamethasone, 50 μg/mL ascorbate 2-phosphate, 40 μg/mL L-proline (Sigma-Aldrich), 1×insulin-transferrin-selenium (ITS, Invitrogen), and 10ng/ml TGF-β3 (R&D Systems, Minneapolis, MN)).
Cell viability was assessed after 8 weeks of culture with a Live/Dead Viability/Cytotoxicity kit (Invitrogen) and observed by an epifluorescence microscopy (CKX 41, Olympus). Four fields (720µm×533µm for each) per sample were analyzed. Percentage of live cells was calculated based on the number of green stained cells divided by the total number of cells (green and red stained cells, dual stained counted once as dead). The total live cell number was further estimated using the CellTiter 96 AQueous Cell Proliferation Assay (Promega, Madison, WI) after 8 weeks of culture. The dimension of constructs was also measured with a caliper.

Levels of biologically active type I IFN in serum were determined using an encephalomyocarditis virus (EMCV) L929 cell cytopathic effect bioassay as described previously (Sheehan et al., 2006 (link); Sheehan et al., 2015 (link)). Briefly, serial dilutions of sera in DMEM containing 10% FBS were applied in duplicate to L929 cells (2 × 10⁴ cells/well) in 96-well plates. Following incubation for 14 hours at 37°C, cells were infected with EMCV diluted in DMEM containing 2% FBS at a MOI of 7. At 8 hours post-infection, IFN-mediated protection was assayed using CellTiter 96 aqueous cell proliferation assay as per the manufacturer’s protocol (Promega). IFN levels were calculated from an IFN-β standard curve. To ensure protection from EMCV-induced death was due to type I IFN activity, L929 cells also were pretreated with anti-Ifnar1 mAb (MAR-1–5A3, 50 ug/mL) prior to the addition of serum samples.

Cell viability was measured using the CellTiter 96 AQueous Cell Proliferation Assay (Promega) according to the manufacturer’s instructions.

Cytotoxicity toward MCL cell lines in vitro was measured by using the CellTiter 96 Aqueous Cell Proliferation Assay (Promega). Briefly, Mino or HBL2 cells were distributed in a 96-well flat bottom plate (Corning) at a density of 5×10⁴ cells/well. Subsequently, compound 1a (0.001-1,000 nM) or Fcμ-Sec protein selectively conjugated to compound 1b (1-100 nM) were added to the cells. In parallel, unconjugated Fcμ-Sec protein (1-100 nM) was also tested. The cells were exposed to the compound 1a and 1b reagents for 1 h at 37°C, washed three times with RPMI 1640 medium, and incubated for an additional 71 h at 37°C. After adding 20 μL Assay Reagent to each well, the cells were further cultured for 3 h at 37°C and the absorbance at 490 nm was measured using a SpectraMax M5 microplate reader with SoftMax Pro software. Data were computed as mean ± SD of triplicates.