Anti cd51 pe

Cells were stained with the following antibodies (all from eBioscience, except where stated); anti-CD4-BV650 (BioLegend, San Diego, CA, USA), anti-Foxp3-(ef450/FITC), anti-CD11b-AF700 (Biolegend), anti-αv-PE, anti-CD44-APC-Cy7, anti-CD62L-PerCP-Cy5.5, anti-Ki-67-PE-Cy7, anti-KLRG1-ef450, anti-CD45.1-APC, anti-CD51-PE, anti-GM-CSF-PE, anti-IFN-γ-APC, anti-IL-17-PerCPCy5.5. Rat IgG2a and rat IgG1, conjugated to respective fluorophores, were used as isotype control antibodies. For intracellular cytokine staining, cells were re-stimulated ex vivo with 20 µg/ml pOVA overnight, and Brefeldin A added for the last 4 h of incubation. Samples were stained with a fixable viability marker (conjugated with eFluor455, eBioscience) prior to surface staining. For subsequent intracellular antigen staining, samples were washed once in FACS buffer (PBS, 2% FCS, 0.01% NaN₃) and then processed according to manufacturer’s instructions (eBioscience for transcription factors, BD for intracellular cytokines). Flow cytometric data were acquired using a BD LSR Fortessa cell analyzer (BD) and data analyzed using FlowJo software (Treestar version 3.2.1, Ashland, OR, USA).

Measurements were performed on a FACS Aria II (BD Biosciences, San Jose, CA, USA) and SH800 cell sorter (Sony, Tokyo, Japan), then analyzed using FlowJo software (TreeStar, Ashland, OR, USA). Single-cell suspensions were blocked with anti-CD16/32 antibody (BD Biosciences) for 10 min, followed by staining using flow cytometry (FACS) buffer (1× PBS, 4% heat-inactivated FCS, and 2 mM EDTA) for 15 min. The antibodies for flow cytometry were as follows: anti-CD45-APC (1:50, 103112, BioLegend, San Diego, CA, USA), anti-CD45-FITC (1:50, 103108, BioLegend), anti-CD31-FITC (1:50, 102405, BioLegend), anti-F4/80-PE/Cy7 (1:50, 123113, BioLegend), anti-Ly6C-APC (1:50, 560595, BD Biosciences), anti-Ly6G-BV421 (1:50, 127627, BioLegend), anti-Sca1-APC (1:50, 108111, BioLegend), anti-CD51-PE (1:100, 104105, BioLegend), Streptavidin-PE (1:100, 405203, BioLegend), and Streptavidin-FITC (1:50, 405201, BioLegend), Annexin V-FITC (1:50, 640905, BioLegend), and 7AAD (559525, BD Pharmingen, San Diego, CA, USA).