Cell separation columns

MEC1 cells were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DMSZ, Germany) and cultured in RPMI 1640 medium (Lonza) supplemented with 10% fetal bovine serum, 2 mM glutamine and 15 μg/ml gentamicin (Sigma Aldrich). MEC1 cells stably expressing green fluorescent protein were generated as described previously [23 (link)] and cultured in supplemented RPMI 1640 medium, as above, containing 5 μg/ml blasticidin (Life Technologies). Human umbilical vein endothelial cells (HUVECs; Lonza, Switzerland; passage 5 to 8) were cultured in EGM™-2 BulletKit™ (EBM-2 basal culture medium with 2% FBS plus SingleQuots™ of growth supplements (Lonza)).
Human leukemic lymphocytes were isolated from the peripheral blood of CLL patients, using the human B-cell enrichment cocktail (RosetteSep, StemCell Technologies) or cell separation columns (Miltenyi Biotec). CD19 and CD5 co-expression on cell surface was checked by flow cytometry (FC500, Beckman Coulter) and the purity was always >97%.

Lymphoid cells were obtained from peripheral lymph nodes (see above) and were labeled with 2 µM Oregon Green 488 dye (Invitrogen) for 15 min at room temperature and then cultured in 96 well plates (1.5×10⁵ cells/well) and stimulated for 72 h with 7.5×10⁴ anti-CD3/anti-CD28 mAb-coated T cell expander beads (Invitrogen) or restimulated with MOG_35–55 (25 µg/ml). Cells were labeled with anti-CD4-PE mAb (eBioscience) and proliferation analyzed by two color flow cytometry on a FACSCaliber flow cytometer with BD CellQuest software (BD Biosciences). Additional experiments were carried out by combining T cells with B20-4.1.1 (100 µg/ml), K(1-3) (5 µg/ml) or IgG control (100 µg/ml). In these experiments, the lymphoid cells were further purified into CD3⁺ T cells by passage through cell separation columns (Miltenyi Biotec). Cells were either naive T cells from wild type mice, or T cells taken from mice with untreated EAE and restimulated by culture with BMDCs (3.2×10³/well) and MOG_35–55 (25 µg/ml). T cells were labeled with Oregon Green to assess proliferation. Labeling with Oregon Green was carried out prior to combining the T cells with BMDCs in order to exclude BMDCs from the analysis based on their lack of fluorescence.