Preparation of cell culture

HCT 116 and CCD-18co were obtained from American Type Culture Collection (ATCC) (Rockville, MD USA). HCT 116 cell line (ATCC Number: CCL-247™) was cultured in McCoy 5A media (1x) (Sigma Aldrich, USA) whereas the normal human colon cell line, CCD-18co (ATCC Number: CRL-1790™) was cultured in EMEM (Eagle’s Minimum Essential Medium) (1x) (Sigma-Aldrich, USA). Culturing of HCT 116 and CCD-18co were carried out in a sterile laminar flow chamber to avoid any possible contamination. McCoy 5A and CCD-18co media were enriched with 10 % fetal bovine serum. All incubations in this study were done at a high humidity environment of 5 % carbon dioxide (CO₂) and at a temperature of 37 °C. The cultured cells were observed and checked daily by using an inversion microscope to see the morphology and cell growth, cultured up to 70-90 % confluence of cells. To subculture the cells, the old media was removed from the flask and phosphate buffer saline (PBS) was used to rinse the excess media. Solution of trypsin-EDTA (0.25 % trypsin/0.03 % EDTA) was added to remove the cells from the surface of the tissue culture flask. Media was added to inactivate the trypsin solution and centrifuged at 1000 rpm for 3 min. The cells were collected and transferred to a new labelled flask with a fresh media. Subculture of cells was done every 2 to 3 days for HCT 116 cell line and 4 to 5 days for CCD-18co cells.