H0887

The MIN6 cells (passage 5–25; mycoplasma negative) (kindly provided by Endocrinology, Diabetes and Nutrition Unit, University of Louvain, Ottignies-Louvain-la-Neuve, Belgium) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (Gibco, A3161002C, Rockville, MD, USA), 10 mM of HEPES (Sigma, H0887, Saint Louis, MI, USA), and 1% P/S at 37 °C, 5% CO₂.

U87 and U251 glioblastoma cell lines (identity of cell lines was verified by karyotyping and STR profiling) were obtained from ECACC and were cultured in DMEM cell medium (DMEM-HA, Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 10% fetal bovine serum (FBS) (S0615, Sigma, Dreieich, Germany), 1% penicillin/streptomycin (2321115, Gibco, Carlsbad, CA, USA), 1% Non-essential amino acids (NEAA) (11140050, Gibco, Carlsbad, USA) and 1% Sodium pyruvate (NPY-B, Capricorn Scientific, Ebsdorfergrund, Germany). Glioblastoma stem-like cells were isolated as described previously [12 (link)]. Cells were cultured in DMEM/F12 medium (DMEM-12-A, Capricorn Scientific, Ebsdorfergrund, Germany) including 2% B27 supplement (117504044, Gibco, Carlsbad, USA), 1% amphotericin (152290026, Gibco, Carlsbad, USA), 0.5% HEPES (H0887, Sigma, Dreieich, Germany) and 0.1% gentamycin (A2712, Biochrom, Berlin, Germany) with EGF (100-18B, Peprotech, Hamburg, Germany) and bFGF (315-09, Peprotech, Hamburg, Germany) in a concentration of 20 ng/mL, respectively. All cells were incubated at 37 °C under 5% CO₂ humidified incubator.

Splenocyte suspensions were prepared by grinding spleens of EAE mice through a 70-μm nylon cell strainer at 34 dpi. Splenocytes were seeded at 2 × 10⁵ cells per well in X-VIVO^TM 15 medium (BE02-060F, Lonza, Basel, Switzerland) supplemented with 1% v/v L-glutamine (X0550, Biowest, Nuaillé, France), 0.4% v/v penicillin-streptomycin (L0022, Biowest), 0.1 M HEPES (H0887, Sigma-Aldrich), and 6 μM 2-β-mercaptoethanol (M3148, Sigma-Aldrich) within 96-well plates. Splenocyte cultures were stimulated with 5 μg/mL MOG_35–55 or 5 μg/mL phytohaemagglutinin-L (PHA-L, L2769, Sigma-Aldrich) and compared to non-stimulated (control) condition. After 54 h in vitro, 75 μL of supernatant were harvested and stored at −80 °C to further assess cytokine secretion. At the same time, cell cultures were again supplemented with completed medium containing 1 μCi of [³H]-thymidine per well (NET027Z, PerkinElmer, Waltham, MA, USA). Splenocyte cultures were maintained under the same conditions for an additional 18 h, and incorporated radioactivity was measured in a beta-scintillation counter (Wallac, Turku, Finland). Five replicates per condition (control, MOG_35–55, and PHA-L) and mouse were analysed and the results are shown as the stimulation index. Stimulation indices were calculated by dividing the mean counts per minute (cpm) of MOG_35–55 or PHA-L condition by the mean cpm of the control condition.

Ten μl of sample was incubated at RT on a 200 mesh formvar/carbon grid. Grids were then washed 5 x 1 min in 200 mM HEPES (Sigma-Aldrich H0887) and transferred to 20 μl 1% Uranyl acetate, (UA) (Agar scientific AGR1260A) and incubated for 1 min at RT. Excess 1% UA was removed and the grids were left to dry before imaging. Images were acquired using a 120 kV Tecnai G2 Spirit BioTwin (FEI company) with an Orius CCD camera (Gatan Inc.)