Apc mouse igg1 isotype control

Chicken anti-GFP antibody (Abcam, ab13970, 1:500), Rabbit anti-Prospc (Millipore, ab3786, 1:500), Rat anti-Ki67 (ebioscience, 14-5698-82, 1:200), Rat anti-F4/80 (BioRad, MCA497GA, 1:200), APC anti-mouse CD326 (BioLegend, catalog#118214), PE/Cyanine7 anti-mouse CD45 (BioLegend, catalog#103114), PE/Cyanine7 anti-mouse CD31 (BioLegend, catalog#102418), PE anti-mouse F4/80 (BioLegend, catalog#123110), APC anti-mouse CD11c (BioLegend, catalog#117310), PerCP-Cyanine5.5 anti-human/mouse CD11b (Tonbo, catalog#65-0112), PE anti-mouse FOXP3 (BD biosciences, catalog#560408), APC anti-mouse CD4 (BD biosciences, catalog#553051), FITC Mouse IgG1 isotype control (BD biosciences, catalog#555748), PE Mouse IgG1 isotype control (BD biosciences, catalog#555749), APC Mouse IgG1 isotype control (BD biosciences, catalog#555751), FITC anti-human CD90 (BD biosciences, catalog#555595), PE anti-human CD73 (BD biosciences, catalog#550257), APC anti-human CD105 (BD biosciences, catalog#562408), FITC anti-human CD45 (BD biosciences, catalog#555482), PE anti-human CD34 (BD biosciences, 550761), Alexa Fluor 488 Donkey anti Chicken (Jackson Immuno Research, catalog#703-545-155), Alexa Fluor Cy3 Donkey anti Rat (Jackson Immuno Research, catalog#712-165-153), and Alexa Fluor 488 Donkey anti Rabbit (Jackson Immuno Research, catalog#711-545-152).

Immune activation, senescence and maturation subsets of peripheral blood T-lymphocytes were evaluated by flow cytometry using heparin whole blood direct staining. Briefly, 50μl of heparinized blood was incubated with fluorochrome-conjugated monoclonal antibodies for 30 minutes at 4°C in the dark. After red blood cells lysis, cells were washed and analyzed on a MACS Quant flow cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany). The anti-human monoclonal antibodies used were: PacificBlue-CD3 (clone HIT3a), APC/Cy7-CD4 (clone RPA-T4), PE/Cy7-CD8 (clone SK1), FITC-CD28 (clone CD28.2), APC-CD38 (clone HIT2), PacificBlue-CD45 (clone HI30), PE-CD45RO (clone UCHL1), PE-CD57 (clone HCD57) and PerCp/Cy5.5-HLA-DR (clone L243) from Biolegend (Franklin Lakes, New Jersey, USA); PerCp-CD3 (clone SK7), FITC-CD27 (clone M-T271), APC-CD49d (clone 9F10) and APC Mouse IgG1-isotype Control from BD Biosciences (San Jose, California, USA). The anti-CD49d antibody used in these experiments was not impacted by natalizumab binding [20 (link)]. CD4 and CD8 Maturation subsets were defined according to CD45RO and CD27 expression as naive (N: CD27⁺CD45RO^-), central memory (CM: CD27⁺CD45RO⁺), effector memory (EM: CD27^-CD45RO⁺), effector (E: CD27^-CD45RO^-) and (only for CD8) intermediate (I: CD27^lowCD45RO^-) [21 (link)]. The gating strategy is shown in Fig 1.