Cd62l pecy5

Splenocytes were prepared as described above. Cells were then incubated with the appropriate antibody at 4°C for 30 min in the dark in PBS with 2% FBS. Flow cytometry was performed using a FACS Fortessa II flow cytometer (BD Biosciences), according to standard techniques. For the characterization of splenic cells, the monoclonal antibodies used were: CD3-AF700 (17A2, #100216), CD4-BV510 (GK1.5, #100449), CD8-AF647 (53-6.7, #100727), CD25-PE conjugated (PC61.5, #12-0251-81), B220-BV421 (RA3-6B2, #562922), CD62l-PeCy5 (MEL-14, #104410), CD44-BV605 (IM7, #103047), CD11b-PerCPcy5.5 (M1/70, #101228), CD80-PE (16-10A1, #09605B), F4/80-BV711 (BM8, #123147), CD86-FITC (GL-1, #105005), CD206-AF647 (MR5D3, #565250), CD43-BV421 (S7, #562958), CD11c-AF700 (N418, #117320), MHCII-EFluor480 (M5/114.15.2, #48-5321-82) from eBiosciences (Thermo Fisher Scientific, Villebon-Sur-Yvette, France). Data were analyzed with FlowJo software (Tree Star, Ashland, OR).

Venous blood was drawn from GCA patients into heparin-containing tubes. Whole blood immunophenotyping was performed using 7-Color Immunophenotyping kit with the following antibodies (Miltenyi Biotec, catalog #130-098-456): CD14-FITC (clone Tük4), CD56-PE (clone REA196), CD16-PE (clone REA423), CD4-PerCP (clone VIT4), CD19-PE-Vio® 770 (clone LT19), CD3-APC (clone BW264/56), CD8-APC-Vio 770 (clone BW135/80), CD45-VioBlue® (clone 5B1). Briefly, 100 μl of whole blood was incubated with 10 μl immunophenotyping reagent for 10 min in the dark, at 4°C. After incubation, whole blood was lysed using Red Blood Lysing Solution (Miltenyi Biotec, catalog #130-098-456). Neutrophil phenotyping was performed in 50 μl of whole blood, incubated for 30 min at 4°C in the dark, with the following antibodies (eBioscience): CD16-PE (clone eBioCB16; catalog #50-112-4738), CD62L-PE-Cy5 (clone DREG56; catalog #50-140-71) and CD11b-APC (clone ICRF44; catalog #17-0118-42). After incubation, samples were lysed, using Whole Blood Lysing Reagent Kit (Beckman Coulter; catalog #6602764). All samples were analyzed using flow cytometer MACSQuant Analyzer 10 (Miltenyi Biotec). Analysis of flow cytometry data was performed using MACSQuantify (Analysis Software version 2.8, Miltenyi Biotec) and FlowLogic (Flow Cytometry Analysis Package, version 7.00.0a, Invasion Software Technologies Pvt Ltd).