Celltiter 96 aqueous one solution cell proliferation assay system

After 24 h of culture, β-gal activity was quantified by adding 4-MUG mix (50 mM Tris–HCl, pH 8, 100 mM β-mercaptoethanol, 0.05% Triton X-100, 5 mM 4-methylumbelliferyl beta-d-galactoside) to cells. Fluorescence associated with the reaction product was monitored 24 h after adding the 4-MUG mix using a Cytofluor-II plate reader (Applied Biosystems) with excitation/emission filters at 360/460 nm. Cytotoxicity analysis was performed under similar conditions without virus and measured with the CellTiter 96® AQueous One Solution Cell Proliferation Assay System (Promega). Porphyrins are known to be fluorescent which could interfere with this test. However, the gold-porphyrins used here are not fluorescent. There is no interference with the reading of the 4-MUG test.

For cell proliferation assays, the CellTiter 96 AQueous One Solution Cell Proliferation Assay System (Promega, Madison, WI, USA) was used according to the manufacturer’s instructions. PC12 cells were plated in each well of a 96-well plate and the indicated concentrations of Wnt3a, sclerostin, XAV939, BMP2, BMP4, and BMP7 was added. After 24-hour incubation in a humidified 5% CO₂ atmosphere, PC12 cells were incubated with 20 μL of CellTiter 96 AQueous One Solution reagent per well. The absorbance at 490 nm was measured using a 96-well plate reader (Bio-Rad, Hercules, CA, USA, Model 680 XR).

Cell proliferation assay was performed using the CellTiter 96 AQueous One Solution Cell Proliferation Assay System (Promega, # G3582). U2OS cells were seeded in 96‐well plates (1,500 cells/well) in three technical replicates. Absorbance at 490 nm was measured with the Infinite M1000 Pro Tecan Plate Reader every 12 h for 72 h after 4 h of incubation with the MTS reagent. To determine the doubling time, background absorbance from the wells with medium only was subtracted from that of sample wells and background‐corrected mean absorbance value for each condition was determined. Doubling time was determined using the following formula: doubling time = 72 h*log₂/log(background‐corrected mean absorbance at 72 h) − log(background‐corrected mean absorbance at 0 h). Estimated doubling times were 35 h for U2OS RPB1‐52R and 42 h for U2OS RPB1‐25R cells.

For the MTS assay, 2×10⁵ 3T3-L1 cells were seeded in each of the wells of a 24-well plate and fed with culture medium overnight. A porous polyester Transwell™ insert with a pore size of 0.4 µm was placed in each well of the 24-well plate. Then, tested hydrogels were placed into the Transwell™ insert. The 3T3-L1 cells were then exposed to test hydrogels or laser wavelength of 520 nm (1.0 mW), 808 nm (1.5 mW), or 980 nm (1.5 mW) for different periods of irradiation for 24, 48, or 72 h incubation. The CellTiter 96^® AQueous One solution cell proliferation assay system (Promega, Madison, WI, USA) was used to evaluate cell proliferation and observation of the optical density (OD) of formazan at 490 nm, quantified cell viability. The reagent contained a tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and inner salt (MTS), and the reduction of MTS achieved by untreated cells was set at 100%, and that of test cells was expressed as a percentage of untreated cells.14 (link)–16 (link) Data are shown as the mean ± the standard deviation for six independent experiments.