We filled the tissue compartment of the bMFA with chemotactic, N-Formylmethionine-leucyl-phenylalanine (1 μM; Sigma Aldrich) prior to the experiments. The fixed flow rate at 1 μL/min injects 5000 Carboxyfluorescein Diacetate, Succinimidyl Ester probe labeled neutrophils per minute, at 37°C. With a previously developed computational fluid dynamics (CFD)-based model [23 (link)], we calculated shear rates in different channels of the network. We recorded video clips at 30 fps using a Rolera Bolt camera (QImaging, Surrey, BC, Canada). After 10 minutes of flowing neutrophils into the bMFA, we injected PBS from the inlet port for 5 minutes to completely wash off unbound neutrophils. We scanned the entire bMFA at the 10× objective using an automated stage on an epifluorescence microscope (Nikon Eclipse TE200, Melville, NY, USA). We processed the acquired images and videos using Nikon Elements software.
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