Ht29 lucia ahr cells

The AhR activation was evaluated using three human AhR-reporter gene cell lines (AhR_T47D, AhR_HepG2, AhR_HT29-Lucia) derived from mammary, hepatic, and intestinal tissues, respectively. The AhR_T47D and AhR_HepG2 cell lines used are two home-made stably transfected cells [36 (link)]. AhR_T47D cells were grown in DMEM (Dulbecco’s Modified Essential Medium), and AhR_HepG2 cells were grown in MEM (Minimum Essential Medium). Both culture media were supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 100 μg/mL of an antibiotic mixture of Penicillin-Streptomycin (Pen/Strep).
Colon adenocarcinoma AhR_HT29-Lucia cells were obtained from Invivogen (Toulouse, France) and cultured according to the provider’s instructions in DMEM supplemented with 4.5 g/L glucose, 2 mM L-glutamine, 10% of FBS, and antibiotics including: 100 μg/mL Pen/Strep, 100 μg/mL of Normocin, and 100 μg/mL of Zeocin.
All cell lines were incubated in 75 cm² culture flasks at 37 °C in 5% CO₂. The growth medium was regularly renewed, and weekly passages upon 80–90% confluency were conducted by rinsing cell layers with phosphate-buffered saline solution (PBS, 1X, pH 7.4) and gently detaching the cells using a 0.25% solution of trypsin-EDTA.

AhR_HT29-lucia™ cells were obtained from Invivogen (Toulouse, France) and maintained as suggested by the providers in the Growth Medium: DMEM, 4.5 g/L glucose, 2 mM L-glutamine, 10% (v/v) heat-inactivated fetal bovine serum (FBS), Penicillin-Streptomycin (100 U/mL–100 μg/mL), 100 μg/mL Normocin™, 100 μg/mL Zeocin™. For AhR reporter gene assays, Assay Medium was used as follows: DMEM, 4.5 g/L glucose, 2 mM L-glutamine, and 10% FBS. Culture conditions were 37 °C and humidified atmosphere (5% CO₂) incubation.
For measuring the AhR induction caused by fermentation supernatants from the M-batches, cells were seeded at 2.5 × 10⁵ cells/mL in clear 96-well plates (100 μL/well) and incubated overnight to allow attachment. Confluence (80–90%) was verified under the microscope, and treatments (20 μL of filtered fermentation sample + 80 μL of cell assay medium) were added to complete a final volume of 200 μL/well. The blank controls used included cells treated with the Assay Medium only and cells treated with the fermentation’s nutritional medium (M-SHIME medium). A positive control of AhR activation (FICZ) was included in all plates.