Hisep media

The human breast cancer cell line, MCF-7, and human cervical carcinoma cell line, HeLa were maintained in DMEM (Sigma, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma, USA) and 100x Pen-strep (Sigma, USA) in a humidified atmosphere of 5% CO₂ in air at 37°C. Lymphocytes were isolated from healthy non-smoking donors using HiSep Media (HiMedia, India) as per the manufacturer's instructions [23 ] and were maintained in RPMI media (Sigma, USA).

Approximately 10000 HeLa cells/well were plated in 96-well plate and incubated for 24 h. After attachment, the cells were treated with different concentrations of quercetin ranging from 1 to 150 µM for 24 and 48 h. Similarly, cells were treated with vehicle control using DMSO. Morphological changes in HeLa cells were recorded using an inverted microscope (Labomed, U.S.A.). Following the treatment, MTT (Sigma–Aldrich) at final concentration of 0.5 mg/ml was added and incubated at 37°C for 2 h. The formazan crystals were solubilized with 100 µl of DMSO with 20-min incubation at 37°C (Sigma–Aldrich). Absorbance Microplate Reader (BioTek, U.S.A) was used to record the absorbance at 570 nm and calculate the viability of the cells. The experiments were repeated thrice and expressed as an average. The cell viability was calculated following the below-mentioned formula:

Lymphocytes were isolated from fresh blood using HiSep Media (HiMedia, India) following the manufacturer’s instructions. They were then resuspended in RPMI media and plated in 96-well microplates at approximately 10,000 cells/well and treated with quercetin as stated above. MTT assay was performed after 24 h exposure.