μquant universal microplate spectrophotometer

Cells were transfected with the indicated plasmids for 24 h and seeded into 96-well plates at a density of 3 × 10³ cells/well. At 48 h, 72 h, 96 h after transfection, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was measured to determine cell viability. The absorbance at 570 nm was measured using a μQuant Universal Microplate Spectrophotometer (Bio-Tek Instruments, Winooski, VT, USA). For the colony formation assays, the cells were counted and seeded in 12-well plates at a density of either 3 × 10² cells/well (Huh7, QGY-7703) or 1 × 10³ cells/well (HepG2). The culture medium was changed every 3 days. When the number of colonies contained more than 50 cells, the cells were washed with 1 × PBS and they were stained by crystal violet.

Cell viability was evaluated using MTT assay. In total, 1 × 10⁴ cells were placed and cultured in a well of a 96-well microplate overnight to achieve firm attachment and then treated with platinum-containing chemotherapeutic agents either CDDP or TFBPC at the concentrations of 0.625, 1.25, 2.5, 5, 10, 20, 40 or 80 μM for another 48 h. A viable cell count was determined by adding MTT reagent (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; SERVA, Heidelberg, Germany) at a concentration of 5 mg/mL in PBS followed by measurement of the absorbance at 570 nm for each well detected by μQuant™ universal microplate spectrophotometer (BioTek, Winowski, VT, USA). The assessment procedures were followed under the manufacturer’s instructions. The final results were represented as mean values in the same experimental group detected by a microplate reader.

To determine the dose response of drug-resistant PC3M1G4 cell lines, PC3M and v6A3 cells were seeded in triplicate in 96-well plates at 5000 cells per well and incubated for 24 h. A range of concentrations (1000, 100, 10, 1, 0.1, 0.01, 0.001, 0.0001 nM) of DOX in fresh medium were added to the cells. Control cells were treated with appropriate volumes of fresh medium. After 48 h, 20 μl of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (5 mg/ ml) (Sigma-Aldrich, St. Louis, MO) was added to each well, followed by incubation at 37°C in 5% CO₂ for 4 h. Subsequently, 100 μl of DMSO (Sigma-Aldrich, St. Louis, MO) was added and the plate was shaken for 20 min at RT to dissolve the formazan crystals. The optical density (absorbance units) was read at a wavelength of 562 nm on a μQuant Universal Microplate Spectrophotometer (Bio-Tek Instruments, Winooski, VT). The growth inhibition curve was generated using GraphPad Prism 6.0 software (GraphPad Software Inc., San Diego, CA). Absolute IC50 values were calculated using the log inhibitor vs. response curves for nonlinear regression.

An ELISA was performed to analyze the binding of synthesized biotinylated peptides to recombinant CD44-Fc or CD44v6-Fc. Briefly, 100 μl of recombinant protein at 1 μg/ml in 0.05 M carbonate/bicarbonate coating buffer (pH 9.6) was added to each well of a 96-well microtiter plate and coated for 24 h at 4°C. The plates were blocked with 1% BSA in PBST buffer (0.05% Tween 20 in PBS) and then incubated with 100 μl of peptides or antibodies at various concentrations in 0.5% BSA in PBST for 2 h at RT. After washing extensively for five times with PBST buffer, the plates were incubated with 100 μl of HRP-conjugated streptavidin (1:2000 diluted with 0.5% BSA in PBST buffer) for 1 h at RT. After washing five times with PBST buffer, the Sigma fast O-phenylenediamine dihydrochloride tablets (Sigma-Aldrich, St. Louis, MO) were used as substrates and the absorbance was measured at 490 nm using an endpoint assay on a μQuant Universal Microplate Spectrophotometer (Bio-Tek Instruments, Winooski, VT).

MTT assay was used to evaluate the cell viability as previously described^{60 (link)}. HUVECs were seeded in 96-well plates at a density of 1 × 10⁴ cells/well with EGM2. Four hours later, cells were transfected with 20 nM eIF2α siRNA or scrambled siRNA, and treated with various treatment factors or vehicle, respectively. After 48 h, the absorbance at 570 nm was detected using a μQuant universal microplate spectrophotometer (Bio-tek, Winooski, USA).