Abstract The endothelial blood-brain barrier (BBB) strictly controls immune cell trafficking into the central nervous system (CNS). In neuroinflammatory diseases such as multiple sclerosis, this tight control is, however, disturbed, leading to immune cell infiltration into the CNS. The development of in vitro models of the BBB combined with microfluidic devices has advanced our understanding of the cellular and molecular mechanisms mediating the multi-step T-cell extravasation across the BBB. A major bottleneck of these in vitro studies is the absence of a robust and automated pipeline suitable for analyzing and quantifying the sequential interaction steps of different immune cell subsets with the BBB under physiological flow in vitro. Here we present the Under-Flow Migration Tracker ( UFM Track) framework and a pipeline built based on it to study the entire multi-step extravasation cascade of immune cells across brain microvascular endothelial cells under physiological flow in vitro. UFM Track achieves 90% track reconstruction efficiency and allows for scaling due to the reduction of the analysis cost and by eliminating experimenter bias. This allowed for an in-depth analysis of all behavioral regimes involved in the multi-step immune cell extravasation cascade. The study summarizes how UFM Track can be employed to delineate the interactions of CD4 + and CD8 + T cells with the BBB under physiological flow. We also demonstrate its applicability to the other BBB models, showcasing broader applicability of the developed framework to a range of immune cell-endothelial monolayer interaction studies. The UFM Track framework along with the generated datasets is publicly available in the corresponding repositories. Author summary Immune cells continuously travel through our body to perform immune surveillance. They travel within blood vessels at a very high speed and slow down upon reaching their target organ by the sequential interaction with different adhesion and signaling molecules on the vascular endothelial cells. The study of molecular mechanisms mediating this multi-step extravasation of immune cells has been significantly advanced by in vitro cultures of microvascular endothelial cell monolayers. The dynamic interaction of the immune cells with endothelial monolayers can be imaged over time in vitro in microfluidic devices under physiological flow. The 2-dimensional structure of the endothelial monolayer allows for reliable visualization of the extravasation process required for the study of the molecular mechanisms involved. The manual analysis of the acquired imaging data is time-consuming and prone to experimenter error. Analysis automation is, however, hampered by the similar appearance of the unlabeled immune and endothelial cells and by the flow causing rapid immune cell displacement. Here we introduce UFM Track, the under-flow migration tracker framework allowing for automated analysis of immune cell interactions with microvascular endothelial cells under flow in vitro. UFM Track performs comparably to the manual analysis of an experienced researcher, eliminates experimenter’s bias, and improves the accuracy of the immune cell tracking. Taken together, UFM Track sets the stage for scalability of in vitro live cell imaging studies of immune cell extravasation.

<sup>UFM</sup>Track: Under-Flow Migration Tracker enabling analysis of the entire multi-step immune cell extravasation cascade across the blood-brain barrier in microfluidic devices

UFMTrack: Under-Flow Migration Tracker enabling analysis of the entire multi-step immune cell extravasation cascade across the blood-brain barrier in microfluidic devices