Human monocyte isolation:
Human blood was obtained from healthy donors with informed consent and ethical approval in the form of leukocyte cones purchased from the NHS Blood and Transplant service. Leukocyte cones contain waste leukocytes isolated from individuals donating platelets via apheresis, and consist of a small volume (~10 ml) of packed leukocytes with few red blood cells or platelets. For monocyte isolation, blood was diluted with 1:2 with PBS followed by separation using a Histopaque gradient and centrifugation as previously described (Purvis et al., 2020). Following human peripheral blood mononuclear cell (PBMC) isolation and washing, approximately 1 × 108 PBMCs were labelled and negatively selected using the pan human monocytes isolation kit and MACS separation (Miltenyi Biotec, Bisley, Surrey, UK). Monocytes were resuspended at 4 × 106 cells·ml-1 in chemotaxis buffer (RPMI 1640, 25 mM HEPES, 0.5% (w/v) BSA) and left on ice until required.

ACEA xCELLigence real-time cell migration:

Experiments were carried out with CIM-16 plates and an xCELLigence RTCA-DP instrument (ACEA, San Diego, USA) as previously described (Iqbal et al., 2013). Chemoattractants (complement C5a and CCL2, both purchased from Peprotech,UK) were made to desired concentrations (10 nM) in chemotaxis buffer (RPMI 1640/25 mM HEPES/0.5% (w/v) BSA) and loaded into the lower wells of the CIM-16 plate. Upper wells were filled with chemotaxis buffer and plates equilibrated for 30 min at RT. BMDM or human monocytes were resuspended in chemotaxis buffer and incubated with ibrutinib (1-30 µM) or vehicle (0.3 % DMSO) for 60 min at 37°C, 5% CO2. Cell suspensions (BMDM; 2x106/mL; hMonocytes; 4x106/mL) were placed into the wells of the upper chamber, and the assay performed with recording of cellular impedance every 15 s for up to 8 h.
Macrophage NF-kB reporter cell line (RAW Blue)
A commercial macrophage reporter cell line (RAW Blue cells; InvivoGen, San Diego, CA; RRID:CVCL_X594) was used: Briefly, cells were derived from murine RAW 264.7 macrophages with chromosomal integration of a secreted embryonic alkaline phosphatase (SEAP) reporter construct, induced by NF‐κB and activator protein 1 (AP‐1) transcriptional activation. Cells were grown to 80 % confluence in DMEM containing 4.5 g·L−1 glucose, heat‐inactivated 10 % FBS, 2 mM l‐glutamine, and 200 μg·ml−1 Zeocin antibiotic (InvivoGen) at 37 °C in 5 % CO2. To minimize experimental variability, only cells with fewer than five passages were used. Cells were plated at 0.95 × 105 per well. After experimental treatments, cell supernatants was added to 180 μl of QuantiBlue substrate (InvivoGen) and incubated at 37 °C for 60 min, plate was then read at an OD 655 nm (OD655) on a microplate spectrophotometer (PherastarFSX, BMG Lab, UK).