Immunofluorescent Staining of Cultured Cells

After fixation, cultures in the OrganoPlate were stained for immunofluorescent markers. As described previously, in short, cells were permeabilized using a Triton X-100 solution for 10 min and blocked using a buffer containing FBS, bovine serum albumin, and Tween-20 for 45 min (42 (link)). Primary antibody was incubated for 1–2 hours or overnight, after which secondary antibody was incubated for 1 hour. The following primary antibodies were used to stain fixed cultures: Anti‐VE-Cadherin 1:500 (Abcam, ab33168), anti‐ICAM‐1 1:50 (R&D systems, BBA3), anti-human CD45 (R&D systems, MAB1430). The following secondary antibodies were used to stain fixed cultures: Goat anti‐rabbit IgG (H+L) Alexa Fluor 488 1:250 (Thermo Fischer Scientific, A11008), Goat anti‐mouse IgG (H+L) Alexa Fluor 647 1:250 (Thermo Fischer Scientific, A21428) and CF647 Goat anti‐mouse IgG (H+L) Alexa Fluor 647 1:250 (Biotium, 20040). Nuclei were stained using Hoechst (ThermoFisher, H3570). After staining, the OrganoPlate was transferred to a confocal high content imaging microscope for automated imaging (Micro XLS-C, Molecular Devices). Images were acquired at 10x magnification at 3 µm increments along the height of the microfluidic channel. Analysis was based on Sum Projection (ICAM expression) or Max projection (VE cadherin) images of the top and bottom 10 z-slices.