Hepes buffer solution

Lymphoblastoid cell lines (LCLs–J1209, C0401, C1504) were established and characterized by the Genethon (Evry, France). They were cultured in RPMI 1640 medium (Eurobio Scientific) supplemented with 10% decomplemented FBS (PAN^TM Biotech). Amino acids, vitamins, sodium pyruvate, penicillin/streptomycin and 2 mM L-glutamine were added at 1× concentrations from 100× stock solutions (all from Gibco, ThermoFisher). Four cell lines of DLBCLs, two ABC subtypes (U2932 and OCILy10) and two GCB subtypes (SUDHL4 and SUDHL6), were cultured in RPMI 1640 medium supplemented with 10% decomplemented FBS, pyruvate (1×), penicillin/streptomycin (1×), L-glutamine (1X) and 10 mM of HEPES buffer solution (Gibco ThermoFisher). All cell lines were maintained at 37 °C in a humified 5% CO₂ atmosphere and were mycoplasma-free (MycoAlert Mycoplasma Detection Kit). Samples from healthy subjects were obtained from the University Hospital Center of Limoges after their informed consent.

Murine lung-carcinoma-derived cell line L2 lung-carcinoma-derived fibroblastic cells [40 ], human A549 (human lung carcinoma (ATCC CCL-185, ATCC, Manassas, VA, USA)), and HULEC-5a (human microvascular endothelium (ATCC CRL-3244)) cell lines were used in this study. Cells were cultured in cell culture dishes with Dulbecco’s Modification of Eagle’s Medium (DMEM) (Corning, Corning, NY, USA) containing 4.5 g/L of glucose, L-glutamine, and sodium pyruvate and supplemented with 10% of FBS, 100 U/mL of penicillin, 10 μg/mL of streptomycin (Lonza, Basel, Switzerland), GlutaMax (Gibco, Waltham, MA, USA), a 0.01 M HEPES buffer solution (Gibco), and MEM nonessential amino acids (Corning).

In vitro live cell imaging of MoC interaction with BBB endothelial cells was performed as previously described (21 (link)). M^unpolarized, M^LPS+IFN-γ and M^IL-4+IL-13 macrophages were resuspended in migration assay media (MAM) containing DMEM, 5% FBS, 4mM L-Glutamine (A2916801, Gibco, Paisley, UK) and 25mM HEPES buffer solution (15630-056, Gibco, Paisley, UK) at a concentration of 1 x 10⁶ cells/ml. A total of 2 x 10⁵ cells were used per movie. Accumulation of M^unpolarized, M^LPS+IFN-γ and M^IL-4+IL-13 macrophages on BBB endothelial cells in the flow chamber occurred for an interval of 5 min, at a low shear pressure of 0.1 dyn/cm², followed by an increase in the shear flow at physiological levels of 1.5 dyn/cm² for 25 min. The total recording time was 30 min, with 10 seconds interval between each frame. Image acquisition was performed using the phase contrast at an inverted microscope (AxioObserver, Zeiss, Feldbach, Switzerland) with a 10x objective. The image analysis was performed using Fiji (National Institute of Health, Bethesda, MD, USA). The number of arrested macrophages per condition was assessed at 40 seconds after onset of physiological shear flow.

Culture of human GBM cells (U87-MG, U373) is described above. NHA cells were purchased from Lonza (Slough, UK) and cultured in low-glucose DMEM medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; PAA, Pasching, Austria), 100 U penicillin/streptomycin (PAA), 2 mM L-glutamine (PAA) and 20 nM HEPES buffer solution (Gibco, Life Technologies). U87-MG cells of passages 29, 30 and 31, U373 cells of passages 40, 41 and 42, and NHA cells of passages 2 and 6 were used in the experiments.
Frozen tissue samples from GBM patients were obtained from the Cancer Center Amsterdam, VU University Medical Center Amsterdam, whereas frozen control post-mortem non-malignant brain tissue samples were obtained from the Academic Medical Center of the University of Amsterdam. Local ethics committees waved use of material, as it fell under the Dutch Code of proper secondary use of human tissue. The research was performed on ‘waste’ material, stored in a coded fashion.