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).