Cliona sp. extract, and CH, CD, and CE fractions, were evaluated for their cytotoxic activity in two cell lines against HEB-2 and HCT-116 (Human larynx carcinoma and Colon carcinoma) using MTT assay as explained by [19 (link),74 (link),75 (link)]. Vinblastine sulphate and DMSO (Dimethyl sulfoxide) were used as positive and negative controls, respectively. The tested cell lines were obtained from VACSERA Tissue Culture Unit Giza, Egypt. DMEM (Dulbecco’s Modified Eagle’s Medium) was used for the tested cells’ propagation. Stock solutions of the extract and fractions were prepared in 10% DMSO in ddH2O. The cytotoxicity was determined using the MTT assay as described by [19 (link),75 (link)].
In brief, cells were seeded in 96-well plates (100 µL/well at a density of 1 × 104 cells/mL) and incubated in 5% CO2 at 37 °C for 24 h. Cells were treated in triplicate with various concentrations of the tested extract and fractions after 24 h. The viable cell yield was determined by a colorimetric method as follows: after additional 24 h, the supernatant was removed and a crystal violet solution (1%) was added to each well for at least 30 min. Then, the stain was removed and the plates were washed using tap water until all the excess stains were removed. Next, 30% Glacial acetic acid was added to the whole wells and mixed. The absorbance of the plates was measured after gently shaking the Microplate reader (TECAN, Inc., Morrisville, NC, USA), using a test wavelength of 490 nm. Compared with the untreated cells, the optical density was measured with the microplate reader (SunRise, TECAN, Inc, Morrisville, NC, USA) to determine the viable cell numbers. The following equation was used to determine the percentage of viability.
where ODt is the mean optical density of wells treated with the tested sample and ODc is the mean optical density of the untreated cells. The relation between the surviving cells and drug concentration was plotted to obtain the survival curve of each tumor cell line after treatment with the specified extract or fraction. The concentration required to cause toxic effects in 50% of the intact cells (IC50) was estimated from the graphic plots of the dose response curve for each concentration using the Graphpad Prism 5 software (Graphpad software, San Diego, CA, USA).
In brief, cells were seeded in 96-well plates (100 µL/well at a density of 1 × 104 cells/mL) and incubated in 5% CO2 at 37 °C for 24 h. Cells were treated in triplicate with various concentrations of the tested extract and fractions after 24 h. The viable cell yield was determined by a colorimetric method as follows: after additional 24 h, the supernatant was removed and a crystal violet solution (1%) was added to each well for at least 30 min. Then, the stain was removed and the plates were washed using tap water until all the excess stains were removed. Next, 30% Glacial acetic acid was added to the whole wells and mixed. The absorbance of the plates was measured after gently shaking the Microplate reader (TECAN, Inc., Morrisville, NC, USA), using a test wavelength of 490 nm. Compared with the untreated cells, the optical density was measured with the microplate reader (SunRise, TECAN, Inc, Morrisville, NC, USA) to determine the viable cell numbers. The following equation was used to determine the percentage of viability.
where ODt is the mean optical density of wells treated with the tested sample and ODc is the mean optical density of the untreated cells. The relation between the surviving cells and drug concentration was plotted to obtain the survival curve of each tumor cell line after treatment with the specified extract or fraction. The concentration required to cause toxic effects in 50% of the intact cells (IC50) was estimated from the graphic plots of the dose response curve for each concentration using the Graphpad Prism 5 software (Graphpad software, San Diego, CA, USA).
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