In vitro cytotoxicity and clonogenicity assays were performed using the sulforhodamine B (SRB) (MP Biomedicals, LLC) protein staining assay [40 ,41 (link),42 (link),43 ], using the cell lines described before. Briefly, cells were seeded in 384- or 96-well microtiter plates at a density of 1 × 104 cells/mL, and placed in an incubator (5% CO2 and 37 °C) for 8 h. Afterward, different concentrations (0.08, 0.4, 2, and 10 µg/mL) of pure compounds and positive controls (POD and VP-16) were added in triplicate and incubated for 72 h.
Afterward, cells were fixed with cold trichloroacetic acid (30% in water) and stained with SRB (0.4% in a 1% of acetic acid solution). Cells were washed with 1% of acetic acid solution. Finally, the bound colorant was solubilized with Tris-base to obtain optical density (ODsample). The bound colorant was proportional with either total protein or cells amount. DMSO (final concentration of 0.5%) was used as vehicle and blank (ODblank). The total protein concentration in a single plate with cells at the beginning of the assay was considered as zero (ODzero). Microtiter plates were incubated for 72 h after which the total protein concentration was determined with Equation (1). This assay measures the respective absorbance at 490 nm using a spectrophotometer (Molecular Devices, SPECTRA max plus 384).
To determine the recovery percentage, an in vitro clonogenicity assay was performed. Cells were seeded, incubated, and treated in microtiter plates under the previously described conditions. After 72 h of treatment, the culture media of each well was removed, followed by two washes with phosphate buffered saline (PBS), before warm media supplemented with FBS was added to the cells and incubated for an additional 72 h in the same conditions. The percentage of proliferation was then determined using Equation (1).
We have defined percentage of recovery as the percentage of survival at 72 h of treatment added to the percentage of proliferation calculated 72 h post-treatment. This is outlined in Equation (2) andFigure 4 :
The half inhibitory concentrations of lignans and controls were calculated from six concentrations (0.0032, 0.016, 0.08, 0.4, 2, and 10 µg/mL). Data analysis was performed using the relationship between the % of survival cells (total protein, Equation (1)) and the relative optical density vs. concentration as a logarithm expression. The IC50 values were reported as a mean of three independent experiments ± standard deviation (S.D.).
Afterward, cells were fixed with cold trichloroacetic acid (30% in water) and stained with SRB (0.4% in a 1% of acetic acid solution). Cells were washed with 1% of acetic acid solution. Finally, the bound colorant was solubilized with Tris-base to obtain optical density (ODsample). The bound colorant was proportional with either total protein or cells amount. DMSO (final concentration of 0.5%) was used as vehicle and blank (ODblank). The total protein concentration in a single plate with cells at the beginning of the assay was considered as zero (ODzero). Microtiter plates were incubated for 72 h after which the total protein concentration was determined with Equation (1). This assay measures the respective absorbance at 490 nm using a spectrophotometer (Molecular Devices, SPECTRA max plus 384).
To determine the recovery percentage, an in vitro clonogenicity assay was performed. Cells were seeded, incubated, and treated in microtiter plates under the previously described conditions. After 72 h of treatment, the culture media of each well was removed, followed by two washes with phosphate buffered saline (PBS), before warm media supplemented with FBS was added to the cells and incubated for an additional 72 h in the same conditions. The percentage of proliferation was then determined using Equation (1).
We have defined percentage of recovery as the percentage of survival at 72 h of treatment added to the percentage of proliferation calculated 72 h post-treatment. This is outlined in Equation (2) and
The half inhibitory concentrations of lignans and controls were calculated from six concentrations (0.0032, 0.016, 0.08, 0.4, 2, and 10 µg/mL). Data analysis was performed using the relationship between the % of survival cells (total protein, Equation (1)) and the relative optical density vs. concentration as a logarithm expression. The IC50 values were reported as a mean of three independent experiments ± standard deviation (S.D.).
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