24 well plate transwells

Caco-2 cells were seeded in 24-well plate transwells (0.4 μM pore size, Costar) at 200,000 cells/transwell. For apical/basolateral treatments, 200,000 Caco-2 cells were mixed with 50,000 NCI-H716 cells in 6-well plate transwells (0.4 μM pore size, Costar) to mimic the ratio of epithelial cells to enteroendocrine cells in the human colon⁴⁴ . Media was changed on days 4, 8, 12, 16, and 18 to differentiate Caco-2 cells in vitro⁴⁵ . On day 21, fully differentiated and polarized cells were validated for epithelial integrity by FITC-dextran permeability assay prior to treatment with BAs.

Undifferentiated Caco-2 cells were seeded in 24-well plate transwells (0.4 μM pore size, Costar) at 200,000 cells per transwell. For apical vs basolateral bile acid treatments, 200,000 Caco-2 cells were mixed with 50,000 NCI-H716 cells in 6-well plate transwells (0.4 μM pore size, Costar) to mimic the ratio of epithelial cells to enteroendocrine cells in the human colon (Cristina et al., 1978 (link)). Media was changed on days 4, 8, 12, 16, and 18 to differentiate Caco-2 cells in vitro (Lea, 2015 ). On day 21, fully differentiated and polarized cells were validated for epithelial integrity by FITC-Dextran permeability assay prior to treatment with bile acids. Briefly, differentiated Caco-2 epithelial integrity was assayed by measuring passive diffusion of 4 kDa FITC-Dextran (Sigma Aldrich) added at a concentration of 5 μM to the apical chamber in 100 μL PBS, while the basolateral chamber contained 500 μL PBS. Diffusion from the apical to basolateral side was measured by fluorescence reading in PBS on the basolateral side of the transwell system using a SpectraMax M5 plate reader (Molecular Devices, San Jose, CA) at the ICCB-Longwood Screening Facility at HMS. Fluorescence reading was normalized to the control. Only transwells with a low fluorescence reading were used in transcytosis experiments.