Dulbecco s modified eagle s medium

Aluminium chloride was purchased from Surechem Products, United Kingdom, Nitrobluetetrazolium, Nicotinamide-adenine dinucleotide, ferrozine and Dulbecco's modified eagle's medium (DMEM) were bought from HiMedia laboratories, Mumbai (India). Moreover, quercetin was purchased from Sigma–Aldrich, India and deoxyribose from Fluka Analytical Laboratories, Germany. Fetal bovine serum, l-glutamine and penicillin–streptomycin were purchased from Sigma (USA).

The cells were maintained in a growth medium containing the following constituents: Dulbecco's modified Eagle's medium (Himedia) with 25 mmol/L glucose, 1 mmol/L pyruvate, 4.02 mmol/L L-alanyl-glutamine, and 10% fetal calf serum (Sigma Aldrich). Confluent cells were detached with 0.15% trypsin (Himedia) for 5 min, following which 2 ml of complete medium was added and the cells were centrifuged at 1000 rpm (180 g) for 5 min. Cell suspension was counted using a Neubauer chamber and seeded in 96 well microtiter plates (Himedia) at a density of 1 × 10⁴ cells per well.

Bacterial adhesion with human colon cancer cells was performed (18 ,24 ) with some modifications. The human HT-29 cell line (ATCC no. HTB-38; ATCC) was used, and the cell culture work was carried out at the Central Research Laboratory, SDM College of Medical Sciences and Hospital (Dharwad, India). Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (both from Himedia Laboratories Pvt, Ltd.) were used to grow HT-29 cells in 12-well flat-bottom cell culture plates until they reached 80% confluence. Prior to the experiment, HT-29 cells were washed gently with PBS twice. Subsequently, the selected eight isolates were centrifuged (5,000 x g for 15 min at 4˚C) and suspended in DMEM without antibiotics to provide approximately 10⁹ CFU ml of the bacterial suspension. Additionally, 200 µl of each strain was added to separate wells and incubated for 2 h at 37˚C in 5% CO₂ atmosphere. The HT-29 cells were then washed twice in sterile PBS to remove non-adherent bacteria before being lysed in 2 ml of 0.1% Triton X-100 in PBS. Cell lysates were tenfold serially diluted and plated with MRS agar and incubated for 24 h at 37˚C. The percentage of adherence was expressed using the formula: