Micro xls c high content imaging systems

After the three weeks treatment, the medium of 3D OMS was replaced with new medium containing Calcein-AM (Thermo Fisher Scientific, diluted 1:2000) and Hoechst 33342 (Thermo Fisher Scientific, diluted 1:2000). The plates were incubated for 1 h at 37°C and 5% CO₂. Images were acquired using a confocal microscope Micro XLS-C High Content Imaging Systems (Molecular Devices, US) at 4x magnification.

Staining with CDFDA was performed to visualize active bile canaliculi in the hepatic organoids co-cultured with microvessels. Briefly, a stock solution containing 5 mM 5-(and-6)-carboxy-2′,7′-dichloro-fluorescein diacetate (CDFDA) (Sigma-Aldrich, #2188) was prepared in 100% Dimethyl Sulfoxide (DMSO) (Sigma, #D8418), aliquoted, and stored at -20 °C. For the assay, culturing media were aspirated in all graft chambers and perfusion in- and outlets and 50 µL of Hep-Medium containing 5 µM CDFDA (1:1000), and Hoechst (Thermo Fisher Scientific, #H3570, 1:2000 in PBS) was added to each well. The OrganoPlate Graft was incubated for 30 min in the incubator (37 °C, 5%, CO2) on an interval rocker switching between a + 14° and − 14° inclination every 8 min (OrganoFlow S, Mimetas). As negative control, Hep-Medium media containing 0.1% DMSO and Hoechst (Thermo Fisher Scientific, #H3570, 1:2000 in PBS) was prepared. At the end of the incubation, chips were washed 6 times with PBS and imaged Micro XLS-C High Content Imaging Systems (Molecular Devices, US).

Images were captured using the ImageXpress Micro XLS (Molecular Devices) and Micro XLS-C High Content Imaging Systems (Molecular Devices) and processed using Fiji^{34 (link)} to enhance contrast and improve visualization. To monitor the integrity of the Caco-2 tubules, phase-contrast images were recorded before and after exposure to enterotoxins. This was routinely performed immediately after measurement of baseline TEER and after the required toxin exposure time. Fixed and stained OrganoPlates were stored at 4 °C until imaging and equilibrated at room temperature at least 30 min before imaging. Maximum intensity projection images were saved as TIFF files after confocal imaging of stained cells.