N formylmethionine leucyl phenylalanine

Femurs were removed and marrow flushed out with complete medium (RPMI, 10% foetal calf serum, 10 units/ml penicillin, 10 μg/ml streptomycin and 2 mM l-glutamine, all Gibco, ThermoFisher, UK). Single-cell suspensions were prepared by passing the cells through a 19 G needle. Neutrophils were isolated using the EasySep^TM Mouse Neutrophil Enrichment Kit (StemCell Technologies, #19762), as per the manufacturer’s guidelines. Neutrophils were activated by 30 min incubation with 100 nM fMLF (N-formylmethionine-leucyl-phenylalanine, Sigma-Aldrich #F3506) and 10 µM cytochalasin B (Sigma-Aldrich #C2743) before being thoroughly washed twice.

The induced mobility or chemotaxis of peripheral blood isolated neutrophils and lymphocytes was carried out following a method previously described (57 (link)). Boyden chambers with two compartments separated by a polycarbonate filter (3 µm of diameter; Millipore, Ireland) were used to evaluate the chemotactic index (CI). Aliquots of 300 µL of neutrophil and lymphocyte suspensions (10⁶ cells/mL) in Hank’s medium were deposited in the upper compartment, and aliquots of 400 µL of the chemoattractant agent N-formylmethionine-leucyl-phenylalanine (Sigma-Aldrich) at a concentration of 10⁻⁸ M in the lower compartment of the chambers. The chambers were incubated for 3 h at 37°C, and the filters were fixed and stained with Giemsa’s solution (Sigma-Aldrich). The number of neutrophils and lymphocytes on the lower face of the filter was counted in 20 microscope fields using an immersion objective (×100) and recorded as CI.

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.