Cd49d fitc clone 9f10

Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation over Lymphoprep (Axis-Shield, Oslo, Norway) according to manual instruction. PBMCs were stimulated by adding mixture of peptides: proteolipid protein (PLP139-151 NH2-HSLGKWLGHPDKF-COOH; pepPLP), myelin oligodendrocyte glycoprotein (MOG35-55 (NH2-MEVGWYRSPFSRVVHLYRNGK-COOH; pepMOG) and myelin basic protein (MBP85-99 NH2-VHFFKNIVTPRTPPP-COOH; pepMBP) (each 25 μg/mL) for 21 h (37°C, 5% CO2 in humidified atmosphere). Following the incubation, cells were washed and labelled with 5 μg/mL CD154-PE (clone 89–76, BD Biosciences, San Jose, CA, USA) and CD49d-FITC (clone 9F10, BD Biosciences, San Jose, CA, USA) mAbs for 30 min at 4 °C. CD49d⁺CD154⁺ population was isolated by FACS sorting procedure using a FACSAria with purity mask mode (BD Biosciences, San Jose, CA, USA). The purity of FACS-sorted CD49d⁺CD154⁺ cell fraction assessed by two-color flow cytometry was ~99.5%.

Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation over Lymphoprep (Axis-Shield, Oslo, Norway) according to manual instruction. PBMCs were stimulated by adding a mixture of peptides: Proteolipid protein (PLP_139-151 NH2-HSLGKWLGHPDKF-COOH; pepPLP), myelin oligodendrocyte glycoprotein (MOG_35-55 (NH2-MEVGWYRSPFSRVVHLYRNGK-COOH; pepMOG), and myelin basic protein (MBP_85-99 NH2-VHFFKNIVTPRTPPP-COOH; pepMBP) (each 25 μg/mL) for 21 h (37 °C, 5% CO₂ in humidified atmosphere). Following the incubation, cells were washed and labelled with 5 μg/mL CD154-PE (clone 89-76, BD Biosciences, San Jose, California, USA) and CD49d-FITC (clone 9F10, BD Biosciences) mAbs for 30 min at 4 °C. CD49d⁺CD154⁺ population was isolated by FACS sorting procedure using a FACSAria with purity mask mode (BD Biosciences). The purity of FACS-sorted CD49d⁺CD154⁺ cell fraction assessed by two-color flow cytometry was ~99.5% (Figure 1D).