Source of reagents and chemicals:
Betanin (Red Beet extract diluted with Dextrin) purchased from TCI Chemicals, streptomycin, penicillin-G, Dulbecco’s Modified Eagle’s Medium (DMEM), L-glutamine, 3-(4,5 dimethylthiozol-2-yl)-2,5-diphenyltetrazoliumbromide, acridine orange (AO), phosphate buffered saline, bovine serum albumin, ethidium bromide (EB), 2’7’diacetyl dichloro fluorescein, ethanol, trypan blue, triton X-100, Rhodamine 123 (Rh-123), dimethyl sulfoxide (DMSO), Dichloro-Dihydro-Fluorescein Diacetate (DCFH-DA) and trypsin-EDTA were bought from Sigma Aldrich chemicals Pvt. Ltd (India). All other chemicals used were of analytical grade, purchased from Hi media laboratories Pvt. Ltd., India.
Cell Culture Maintenance:
Human ovarian (PA-1) cancer cells were obtained from the cell repository, National Centre for Cell Sciences (NCCS), Pune, India. For cell line maintenance DMEM was used and 10% of FBS (Fetal Bovine Serum) was used as a supplement. To prevent any bacterial contamination in the medium, streptomycin (100μg/ml) and penicillin (100 U/ml) were added. Cell lines were then incubated with 5% CO2 in a humidified condition at 37°C.
10 mL of PBS was used to dissolve 50 mg of MTT dye to make stock solution. Using 0.45-micron filter, it was filtered after 1 minute of vortexing. To prevent the exposure of light aluminium foil was used to wrap the apparatus as MTT is light sensitive. Until further use, it was stored at 4°C. To determine betanin cytotoxicity on PA-1 cells, Mosmann method (1983)  was used. Firstly, Haemocytometer was used to count the harvested viable PA-1 cells and DMEM medium was used further. In 96 well plates 1 × 104 cells/ml of density was seeded in each well and to allow attachment it was incubated for 24 hours. After treating the PA-1 cells with control, each well was applied with different concentrations of Betanin (10 - 60 µg/ml). At 37°C PA-1cells were incubated for 24h in a humidified CO2 (5%) and air (95%) incubator. After the incubation period is over the drug containing cells were washed with fresh medium and each well was added with MTT dye (5 mg/ml in PBS) followed by another 4h of incubation at 37°C. The concentrated DMSO (100 µl) was used to dissolve the precipitated purple formazan and using multi-well plate reader the viability of cell was measured at absorbance 540 nm. The results were represented as percentage stable cells in comparison to the control. The values for half maximal inhibitory concentration (IC50) was calculated and the optimum doses were analysed at different time period. From the dose responsive curve of betanin, the values of IC50 were determined where 50% cytotoxicity inhibition was compared to control cells. All experiments were performed in triplicates to minimize errors.
The values were obtained using mean ± SD. One Way Analysis of Variance (ANOVA) and Duncan’s Multiple Range Test (DMRT) were used for statistical comparisons on Statistical Product and Service Solutions (SPSS) software (version 12.0, SPSS Inc. Chicago; http://www.spss.com). The values were taken as statistically significant when p values come under 0.05.
Using the method given by Pereira et al. (1999)  ROS values were generated. PA-1 cells were added in 6 well plates (2 x 106 cells/well) for 24 h before exposing them to different concentrations of the betanin (40 cells/well) for 24 h prior to exposure and different concentrations of the betanin (40 µg/ml) and untreated cells were maintained at 37°C (5% CO2). Overnight grown PA-1 cells were transferred to 24 well plates for 24 hours. After being exposed to 40 µg/ml of betanin, PBS (1%) is used to wash PA-1 cells and loaded with DCFH-DA (25 µM) in DMEM at 37°C for 30 minutes. DMEM was then used to wash the treated cells and fluorescence was recorded for every 5 minutes for the next 30 minutes (emission 535nm, excitation 485nm) through spectrofluorometry at 37◦C. Increase in ROS was calculated by mean slope per min and normalized to the control which is unexposed to betanin.
Measurement of Mitochondrial Membrane Potential (MMP):
Method given by Bhosle, et al. (2005)  was used to measure MMP. For the assay, stock solution was made using 10mg Rh-123dye dissolved in 1ml PBS buffer while working solution was made by taking 4µL of the stock solution and dissolved into 0.96ml PBS. PA-1 cells were exposed to betanin of different concentrations (40µg/ml) and were placed in a six well plate with a cover slip. The cells are kept under incubation for 15 minutes once they are stained with Rh-123. PBS was used to wash and fix the cells. At 535nm wavelength, the intensity of fluorescence was measured and the percentage of PA-1 cells reflecting pathological changes were calculated.
Induction of Apoptosis by betanin:
The method proposed by Baskić et al. (2006)  was used to analyse the apoptotic cell death using fluorescence microscopy. For staining, 200µL of dye mixture was prepared by using 100µL/mg EB and 100µL/mg AO and dissolving it in 1% PBS. A density of 5 x 104 cells/well of PA-1 cells were added in a 6 well plate and given 24hrs of incubation. The cells were detached after 24hr treatment with betanin (40 µg/ml), and then washed using cold PBS. Further, staining was performed using the mixture of AO (100 µg/ml)/EB (100µg/ml) at a ratio 1:1 for 5 min at room temperature. At a magnification of 40x the stained cells were observed using fluorescence microscope. After the treatment finished, the cells were collected and washed with PBS up to three times.
The number of cells showing feature of apoptosis was counted as a function of the total number of cells present in the field.