Spectramax 250 reader

Caspase-3 protease activity was measured by the Caspase-3/CPP32 activity Colorimetric Assay kit (Biovision Incorporate, CA, USA). In brief, cells were seeded in 6-well plates (2x10⁶ cells/well) and treated with drugs for 24 hrs. The cells then were harvested and centrifuged at 4°C for 5 min. The cytosolic extracts were prepared by repeated cycles of freezing and thawing of the cells in 50 μl of lysis buffer. The lysates were centrifuged at 10,000 xg for 5 min. Protein concentrations were determined by the BCA assay kit (Thermo, USA) according to the manufacturer’s instructions. Cell lysates (100 μg) were then diluted with 50μl of lysis buffer followed by addition of 50 μl of the 2X reaction buffer. After incubation in the presence of the fluorescence substrate Ac-DEVD-pNA (200 μM) at 37°C for 2 hrs in dark, the absorbance at 405 nm was determined using a SpectraMax 250 reader (Molecular Devices, CA, USA). For the Annexin V-fluorescein isothiocyanate (FITC)/PI assay, testing cells were washed with cold PBS twice, and then resuspended in the buffer containing Annexin V-FITC and PI. The mixture was kept in dark for 15 min at room temperature before analysis by flow cytometry.

Cell proliferation was measured using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT, Sigma-Aldrich) as previously described [20 (link)]. The optical density (OD) at 550 nm was measured using a 96-well plate SpectraMax 250 reader (Molecular Devices, Sunnyvale, CA, USA). For colony formation assay, cells (150 cells/well for OEC-M1 and 300 cells/well for SCC-15) were seeded into 6-well plates and cultured for 7 days at 37 °C in culture hood. Colonies were fixed, stained with 1% crystal violet, and counted under a microscope.

The OSCC cells were harvested in the exponential growing phase and seeded into 96-well plates (3000 cells/well). After overnight incubation, 200 μl of culture medium containing the test compound was dispensed into each well. Following 72-h treatment, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) dye (Calbiochem, CA, USA) was added. After 2 hrs of incubation, the medium and MTT dye were removed by slow aspiration and 100μl of dimethyl sulfoxide (DMSO) was added to dissolve the remaining MTT formazan crystals. The absorbance at 550 nm was measured using a 96-well plate SpectraMax 250 reader (Molecular Devices, CA, USA).

The cell viability was assessed using the 3-(4,5-diphenyltetrazolium-2-yl)-2,5-diphenyltrazolium bromide (MTT) colorimetric assay. Adherent cells were plated at a density of 5 × 10³ cells/well, and PBMCs were plated at 10⁵ cells/well in 96-well microtiter plates. After 24 and 48 h, solutions with different ESVR concentrations (25-125 μg/mL), diluted in medium with 0.1% DMSO, were added, and medium with only 0.1% DMSO was used as a control. At the end of both periods, 100 μL MTT (0.5 mg/mL) was added to each well. The cell culture was incubated for another 4 h, and 100 μL of DMSO was then added to solubilize the formazan crystals. The absorbance was determined at 570 nm using a SpectraMax 250 reader (Molecular Devices). Cell viability inhibition was calculated using the following formula:
$\begin{array}{c}\text{Cell\hspace{0.17em}viability\hspace{0.17em}}\left(\%\right)=\left[{\text{Abs}}_{\text{treated\hspace{0.17em}cells}}/{\text{Abs}}_{\text{control}}\right]\times 100.\end{array}$