Pancreatic also attain cytotoxicity the percentage of cytotoxicity using

Pancreatic adenocarcinoma cells (PANC-10.05 cells) where bought from ATCC: The global BioSource Centre. The cells were cultured in RPMI 1640 supplemented with 10% FBS, penicillin (100 U/mL) and streptomycin (100 ?g/mL) and maintained at 37°C in the presence of 5% CO2 in a humidified atmosphere.   MTT Assay The inhibition of growth by shikonin will be measured on PANC-10.05 cells will be measured via MTT assay. The cells will be seeded in 96-well plates at a density of 4×103 well.  It will be incubated for 12 hours, thereafter it will be treated with different concentrations of shikonin (1, 3, 10, 30 and 100 ?M). The control group will have dimethyl sulfoxide (DMSO).   After 24-48 hours, the cells will be incubated with MTT (0.25 mg/ml) for 3 hours at 37°C. DMSO will be added until purple formazan crystals are dissolved, and the absorbance of the plate will be read on spectrophotometer. There will be duplicate readings that will be averaged and must subtract the culture medium back from assay reading to give the corrected absorbance. To cell count, the absorbance will be proportional to cell number. To also attain cytotoxicity the percentage of cytotoxicity using the corrected absorbance.   Flow Cytometry Once PANC-10.05 cells (1 × 106 cells) will be harvested and rinsed in cold phosphate buffered saline (PBS). 70% ethanol will be used to fix the cell pellets and then wash in cold PBS, suspended in PI solution (1ml) containing 50 ?g/mL of PI, 0.1% sodium citrate, 0.1% triton X. The samples will be incubated at 4°C in the dark and analysed via the Beckton Dickison FACScan flow cytometer.  The results will be translated on a quantitative scatter plot, indicating the degree of cell apoptosis taking place. JC-1 dye will reveal the cellular intake in the mitochondria. Around 590nm (red fluorescence) the mitochondria are healthy and at 590nm (green fluorescence) the mitochondria are undergoing cell apoptosis.  The quantitative results presented on the graphs will show a relationship of cell growth inhibition and cell apoptosis when shikonin is present. These results are expected to show high levels of cell proliferation inhibition and cell apoptosis. For more conclusive results, this experiment will be repeated over a 3-month period.