Versene

Stable HEK293T NanoLuc VEGFR2 (NLuc-VEGFR2) and HEK293T VEGFR2-HaloTag cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; D6429; Sigma-Aldrich, UK) supplemented with 10% fetal calf serum and incubated at 37 °C/5% CO₂. Cell passaging was performed when cells reached 80% confluency using PBS (Lonza, Switzerland) and trypsin (0.25% w/v in versene; Lonza, Switzerland). We confirmed that these cell lines were mycoplasma free.

VEGF₁₆₅a and VEGF₁₆₅b were obtained from R&D systems (Abingdon, UK). Vandetanib, pazopanib, cediranib and sorafenib were supplied by Sequoia Research Products (Pangbourne, UK). The ONE-Glo™ Luciferase Assay System was obtained from Promega Corporation (Madison, WI, USA). Versene was obtained from Lonza (Basal, Switzerland). G418 was purchased from Life Technologies (Paisley, UK). All other chemicals and reagents were purchased from Sigma-Aldrich (Gillingham, UK).

Jurkat cells (clone E6-1) were maintained in T175 flasks containing RPMI 1640 medium (Lonza) supplemented with 10% fetal calf serum (FCS; Sigma Aldrich) and 2 mM L-glutamine (Sigma Aldrich) at 37°C/5% CO₂. Fresh medium was added to the cells every 2-3 days. Cells were passaged at 70% confluency by withdrawing 2.5 ml of the cells into a fresh T175 flask with medium. HEK293G cells (Glosensor cAMP HEK293, Promega) were grown in T75 flasks containing 25 ml Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma Aldrich, UK) supplemented with 10% FCS at 37°C/5% CO₂. Cells were passaged at 70-80% confluency using phosphate buffered saline (PBS; Lonza) and trypsin (0.25% w/v in versene; Lonza). A mixed population HEK293G cell line was created by transfecting cells with the LgBiT-CXCR4 construct using FuGENE® (Promega) according to the manufacturer’s instructions, followed by selective pressure (1 mg/ml G418) for two to three weeks. HEK293G cells stably expressing the SNAP-CXCR4 construct were kindly gifted from Dr. J. Goulding (University of Nottingham).

Blood samples (20 ml) from clinically affected homozygous NPC1 mutation carriers and healthy donors were collected. Negative selection of peripheral blood monocyte-derived macrophages was performed by incubating full blood for 20 min at RT with RosetteSep Human Monocyte Enrichment Cocktail (StemCell Technologies). An equal volume of washing buffer (D-PBS/2% FBS/1 mM EDTA) was added to each sample and layer of macrophages was separated from red blood cells and plasma by centrifugation on a Ficoll gradient (800 × g for 15 min, GE Healthcare). Potential contamination by red cells was eliminated by incubating cell pellets with ACK lysis buffer (Gibco) for 3 min at RT. Lysis buffer was quenched with 40 ml of washing buffer and cells were centrifuged at 300 × g for 7 min. Cell pellets were resuspended and plated in macrophage complete medium (RPMI 1640/10% FBS/1% PenStrep/1X Pyruvate/1× NEAA) supplemented with 50 ng/ml hM-CSF (Thermo Scientific). After 48 h, 50 ng/ml of fresh hM-CSF was re-added. At 5DIV, media was discarded and adherent cells were washed once in PBS, incubated for 3 min at RT with Versene (Lonza) and scraped in 5 ml macrophage complete medium for further analysis.