Apc cy7 anti cd11b

The infiltrated lymphocytes were isolated as described previously [20] (link). The allografts were cut into pieces and digested with 2 mg/ml collagenase D (Worthington Bio, Lakewood, NJ) combined with 10% fecal calf serum in RPMI 1640 media for 2 h at 37°C. The suspensions were filtered with cell strainers (40 µm, BD Biosciences, San Diego, CA). The isolated cells were stained with FITC-anti-CD4, APC-Cy7-anti-CD11b, PerCP-Cy5.5-anti-IFN-γ, eFlour660-anti-IL-13, PE-anti-IL-17A, APC-anti-Foxp3, and 7-AAD (eBioscience, San Diego, CA) according to eBioscience’s Best Protocols. The cells were assessed with a fluorescence activated cell sorter (FACS) Aria II (BD Biosciences, San Diego, CA) and analyzed using FCS Express 4 Plus (De Novo Software, Los Angeles, CA).

One million leukocytes from each mouse were plated in 96-well plates, centrifuged at 845 x g for 3 min at 4°C and incubated with α-CD16/CD32 (eBioscience/Thermo Fisher Scientific, Waltham, MA, USA) for 20 min at 4°C. Liver and/or spleen leukocytes were there surface-stained for 20 min at 4°C using appropriate combinations of the following fluorochrome-conjugated anti-mouse monoclonal antibodies: FITC or eFluor450anti-TCRγδ (clone GL3), PE anti-NKp46 (clone 29A1.4), PerCP/Cy5.5 anti-CD3ɛ (clone 145-2C11), PE or PE/Cy7 anti-NK1.1 (clone PK136), Alexa Fluor 647 anti-SiglecF (clone ES0-2440), PE/Cy7 anti-Ly6G (clone AE8), FITC or APC/Cy7 anti-CD11b (clone M1/70), Brilliant Violet (BV)421 or BV510 anti-CD4 (clone RM4-5), BV510 or Alexa Fluor 700 anti-CD45 (clone 30-F11), BV605 anti-CD19 (clone 6D5), BV605 anti-Ly6C (clone HK1.4), and BV711 anti-CD8α (clone 53–6.7), plus eFluor 506 or eFluor 780 Fixable Viability Dye (eBioscience, Thermo Fisher Scientific) for dead cell exclusion. All antibodies were from eBioscience (Thermo Fisher Scientific) or Biolegend. Cells were acquired on a BD LSRFortessa or LSRFortessa X-20 (BD Biosciences) and data acquisition and analysis were carried out using the FACSDiva (version 6.2) and FlowJo (version 10.5.3, FlowJo) software packages, respectively.