Siglecf pe vio770

For the evaluation of joint immune infiltration, cells were isolated from the hind paws by digestion in serum-free media containing DNAse I (40µg/ml) and collagenase D (0.5µg/ml) for 1 h. Upon tissue digestion, 30,000 precision counting beads (BioLegend; San Diego, California, USA) were added for total cell quantification and the cell suspension was incubated with live/dead discrimination marker (Fixable Viability eF780, Thermo Fisher Scientific; Waltham, Massachusetts, USA), Fc Receptor blocking reagent (BioLegend) and subsequently stained with an antibody cocktail to characterize the different immune cell populations. The antibodies used were from BioLegend, unless otherwise specified: Ly6C-BV421 (clone HK1.4), CD45-VioGreen (clone 30F11; Miltenyi; Bergisch Gladbach, Germany), MHC-II-BV650 (clone M5/114.15.2), CD11b-BV785 (clone M1/70), B220-FITC (clone RA3-6B2), F4/80-PE (clone BM8), CD3e-PE-Cy5 (clone 145-2C11), SiglecF-PE-Vio770 (clone REA798; Miltenyi), CD11c-APC (clone N418) and Ly6G-AF700 (clone 1A8). A sample containing at least 20,000 beads was acquired in a Fortessa Cytometer (BD; Franklin Lakes, Nueva Jersey, USA) and flow cytometry data was analyzed with FlowJo software v10.4 (ThreeStars).

The differential immune cell count was determined by using either NovoCyte or Beckman Coulter CytoFLEX flow cytometers and analyzed with NovoExpress (Acea Biosciences, USA) or CytExpert software (Beckman Coulter, USA). Differential immune cell counts were performed as previously described (Chan et al. 2016 ). Leukocytes were identified as CD45⁺, alveolar macrophages as CD11c⁺Siglec-F⁺, eosinophils as CD11c⁻Siglec-F⁺, neutrophils as GR-1⁺CD11b⁺ or Ly-6G⁺CD11b⁺, B cells as CD3⁻/CD19⁺ and T cells as CD3⁺/CD19⁻ cells. All antibodies were from Miltenyi Biotech: CD45-VioBlue (130–110-802), CD45-PE (130–110-797), CD64-APC-Vio770 (130–118-685), Siglec-F-PE-Vio770 (130–112-334), Siglec-F-APC (130–102-241), CD11b-PE-Vio615 (130–113-807), CD11b-FITC (130–098-085), CD11c-PE-PerCP700 (130–110-842), CD11c-PE (130–102-799), Ly-6G-PerCP700 (130–117-500), Gr-1-PE (130–112-306), Gr-1-APC (130–102-833), CD3-APC-Vio770 (130–119-793), CD19-PE-Vio770 (130–111-885), Annexin-V-PE (130–118-363), and Viobility 405/520 Fixable Dye (130–109-814).