Fig. 4

Establishing a live imaging setup to capture circulating monocyte transmigration with a ‘trench up’ membrane configuration. (a) Live imaging flow setup, which is comprised of a peristaltic pump, media reservoirs in a 37ºC bead bath, and an incubation stage that holds the device. This setup sits on a Nikon Ti2E microscope to capture real-time phase videos. Schematic of fluidic hToC with circulating monocytes. (b) Comparison of the flow channel geometry between ‘trench down’ and ‘trench up’ membrane configurations. Velocity profiles of an xz slice through the center of the channel, demonstrating a decreased flow velocity within the trench. (c) Schematic illustrating the steps in the monocyte transmigration cascade, which include static adhesion to the endothelium, probing, and transmigration across the barrier. (MCP-1: Monocyte chemoattractant protein-1) (d) The final frame of a 2 h live video of monocyte circulation showing the EC monolayer with monocytes at various stages along the transmigration cascade. Color coding corresponds to the monocyte state along the transmigration cascade based on the phase appearance of the cells. Monocytes that appear phase-bright are on top of the endothelium, cells that are flattening out and probing appear gray, and cells that have transmigrated under the endothelium appear phase-dark.