Foetal calf serum

Ficoll-Paque was from GE Healthcare (Uppsala, Sweden). RPMI-1640 medium (Life Technologies; Carlsbad, CA, USA) was prepared at the Illawarra Health and Medical Research Institute (Wollongong, Australia). Foetal calf serum (FCS) (heat-inactivated before use) was from Bovogen Biologicals (East Keilor, Australia). Dimethyl sulfoxide and trypan blue were from Sigma-Aldrich (St. Louis, MI, USA). Dulbecco’s phosphate-buffered saline (PBS) was from Life Technologies. The Zombie NIR Fixable Viability Kit was from BioLegend (San Diego, CA, USA). Fluorochrome-conjugated monoclonal antibodies (mAbs) (Table 2) were from BD Bioscience (San Jose, CA, USA).

Cell cultures were maintained in R10 medium containing RPMI-1640 media without L-glutamine (Gibco Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% heat-inactivated foetal bovine serum (FBS; Bovogen Biologicals, Christchurch, New Zealand), 10,000 units/mL of penicillin + 10,000 µg/mL of streptomycin (Thermo Fisher Scientific), and 1X GlutaMAX (Thermo Fisher Scientific).
PBMCs were separated from the buffy coats of three healthy donors by standard density gradient centrifugation using Lymphoprep medium (STEMCELL Technologies, Vancouver, Canada) according to the manufacturer’s instructions. Isolated cells were used fresh or cryopreserved in liquid nitrogen vapour phase storage using 90% heat-inactivated foetal calf serum (FCS) (Bovogen Biologicals) plus 10% dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA).
On the day of the experiment, PBMCs were thawed and removed from freezing medium by washing with 10 mL R10 and centrifugation at 500 × g for 5 min. To reduce cell clumping, we resuspended the pellet in 5 mL R10 with 10 µg/mL DNase solution I (STEMCELL Technologies) and incubated it at 37 °C and 5% CO₂ for 1 h. The cells were washed twice to remove DNase. Next, PBMCs were counted using a 0.4% trypan blue staining solution (Thermo Fisher Scientific). The cell density was adjusted as required.

The immortalised monocytic cell line Mono Mac-6 (MM6), a human cell line with characteristics of mature monocytes, was a kind gift from Ziegler-Heitbrock [33] (link) and the monoblastic cell line THP1 was a kind gift from Saunders [34] (link). Both monocytic cell lines were maintained in RPMI medium (Invitrogen) supplemented with 10% heat inactivated foetal calf serum (FCS) (Bovogen) at 37°C in 5% CO₂.
The human brain microvascular endothelial cell line hCMEC/D3 [35] was cultured in endothelial cell basal medium-2 (Lonza) supplemented with 5% FCS, recombinant long R insulin-like growth factor-1 (R-IGF-1), vascular endothelial growth factor, ascorbic acid, hydrocortisone, epidermal growth factor human recombinant and human fibroblast growth factor-B (all from Lonza). The cells were seeded onto 0.3% collagen coated flasks and grown at 37°C in 5% CO₂.