Anti cd49b dx5

Spleens were dissected from mouse spleen using 70µm cell strainers (Falcon) and further processed under sterile conditions. Single-cell suspensions were rinsed-out with RPMI-1640 (Lonza) supplemented with 10% (vol/vol) fetal calf serum (FCS), 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 40 mM β-mercaptoethanol, 100 U ml−1of penicillin, and 100 U ml−1of streptomycin (all reagents from Lonza).
CD4⁺ cells were purified by negative selection with magnetic depletion of B cells, macrophages, DCs, NK cells, granulocytes and CD8⁺ cells using a cocktail of biotinylated antibodies (anti-CD49b, DX5, eBiosciences; anti-GR1, RB6-8C5, produced in house; anti-Ter119, BioXCell; anti-CD11c, N418, produced in house; anti-CD19, BioXCell; anti-CD8β, H35, produced in house; anti-CD25, PC61.5, eBiosciences (used for Tconv-but not Treg- purification); anti-MHCII, M5/114.15.2, eBiosciences). Cells were recuperated after flow-through the magnetic column, with previous incubation with anti-biotin Microbeads (Miltenyi Biotec). Untouched cells were stained to exclude dead cells and incubated with Fc receptor-blocking antibodies CD16/32 (Fc block; BD Pharmingen) and surface staining antibody CD3⁺ and CD4⁺. Tconvs and Tregs were identified in FSC/SSC-low-to-moderate and sorted as GFP^- or GFP⁺ respectively using a BD FACSAria III.

Cell surface staining was performed using the following antibodies: anti-CD11b (M1/70, Miltenyi Biotec), anti-CD11c (N418, BioLegend) anti-CD3 (17A2, eBioscience), anti-CD49b (DX5, eBioscience), anti-CD69 (H1.2 F3, eBioscience), anti-CD86 (GL-1, BioLegend), anti-MHC-II (M5/114.15.2, Miltenyi Biotec), anti-NK1.1 (PK136, BD Bioscience). Dead cells were excluded from analysis (positive for fixable viability dye, eBioscience). For detection of activated T cells splenocytes were stained with anti-CD4 (GK1.5, eBioscience), anti-CD8 (53–6.7, eBioscience), anti-CD43 (1B11, BioLegend) and anti-CD62L (MEL-14, eBioscience). Intracellular IFN-γ (XMG1.2, Miltenyi Biotec), IL-2 (JES6-5H4, Miltenyi Biotec) and TNF-α (MP6-XT22, BioLegend) staining was performed as described [28 (link),29 (link)]. Data were acquired on LSR II flow cytometer (BD Bioscience) and analyses were performed using FACSDiva (BD Bioscience) and Flow Jo (Tree Star, USA) software.