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69949095678
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note
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16,17 using 96-well tissue culture plates. The 100 μl of cell suspension was added to each well of the 96-well tissue culture plate. The cells were allowed to grow in carbon dioxide incubator (37 °C, 5% CO2, 90% RH) for 24 h. Test materials (100 μl) were added after 24 h of incubation to the wells containing cell suspension. The plates were further incubated for 48 h in a carbon dioxide incubator. The cell growth was stopped by gently layering trichloroacetic acid (50%, 50 μl) on top of the medium in all the wells. The plates were incubated at 4 °C for 1 h to fix the cells attached to the bottom of the wells. The liquid of all the wells was gently pipetted out and discarded. The plates were washed five times with distilled water to remove trichloroacetic acid, growth medium, low molecular weight metabolites, serum proteins etc and air-dried. The plates were stained with sulforhodamine B dye (0.4% in 1% acetic acid, 100 μl) for 30 min. The plates were washed five times with 1% acetic acid and then air-dried. The adsorbed dye was dissolved in Tris-HCl Buffer (100 μl, 0.01 M, pH 10.4) and plates were gently stirred for 10 min on a mechanical stirrer. The optical density was recorded on ELISA reader at 540 nm.The cell growth was determined by subtracting mean OD value of respective blank from the mean OD value of experimental set. Percent growth in presence of test material was calculated considering the growth in absence of any test material as 100% and in turn percent growth inhibition in presence of test material was calculated. Adriamycin, 5-FU and paclitaxel were used as positive control.
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69949106658
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note
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3 (402.44): C, 83.57; H, 4.51. Found: C, 83.39; H, 4.42.
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