Cd80 pe cy5

Tumor tissue was digested in a 0.3% collagenase/0.1% hyaluronidase solution, pressed through a nylon mesh filter to obtain a single cell suspension and incubated in red cell lysis buffer (0.17 M Tris-HCl, 0.16 M NH₄Cl) for 3 min, spun down, and resuspended in FACS buffer (PBS + 1.5% FBS). Equal numbers of cells were stained with a viability dye and combinations of the following antibodies: CD8a-PECy7 (eBioscience, San Diego, CA, USA, 25-0081-82), F4/80-PECy7(eBioscience, 25-4801-82), CD206-FITC (Biolegend, 141704), Ly6G-APC (eBioscience, 17-5931-81), CD11b-PE (eBioscience, 12-0112-82), CD45-PE-Cy5 (eBioscience, 15-0451-83), IFN-γ-APC (eBioscience, 17-7311-82), and SIINFEKL pentamer-PE (ProImmune, Oxford, UK). For autophagy analysis using Cyto-ID staining of bone marrow derived macrophages, cells were first stained for surface markers (CD80-PE-Cy5 (eBioscience, 15-0801-81) and CD206-APC (eBioscience, 17-2061-82)) followed by staining with Cyto-ID for 30 min at 37 °C, per the manufacturer’s protocol (Enzo Life Sciences, Farmingdale, NY, USA, ENZ-51031). Flow cytometric data were acquired on a BD FACSCanto II cytometer and analyzed using FACSDiva software version 9.2 (BD Biosciences, San Jose, CA, USA).

BSA and streptavidin were from Sigma-Aldrich. PBS was from Gibco. TT was supplied by the Statens Serum Institut. All recombinant influenza HA subtypes were from Sino Biological: H1N1 HA (A/California/04/2009; H1), H2N2 HA (A/Japan/305/1957; H2), H5N1 HA (A/Vietnam/1194/2004; H5), H3N2 HA (A/Perth/16/2009; H3) and H7N9 HA (A/Shanghai/1/2013; H7). H1N1 HA (A/New Caledonia/20/1999; H1 wt), the HA delta stem mutants H1 I45R/T49R and H1 45glyc, and the human monoclonal antibodies CR6261 and CH65 were provided by D. Lingwood. HIV-1 92BR-gp120 was provided by D.R. Burton (Scripps Institute, San Diego, CA). Human CpG ODN 2006 was from InvivoGen. IL-15 and IL-6 were from PeproTech. Antibodies used were as follows: goat anti–human IgG (FcΥ specific; Jackson ImmunoResearch Laboratories, Inc.); anti–human CD27-APC, anti–human CD38-FITC, CD138-PE, CD19-Percp Cy5.5, CD20-APC, CD80-PE-Cy5, HLA-DR-PE-Cy5, and anti–human κ-Ig biotin (eBioscience); anti-human CD86-Bv510, CCR10-PE, CD62L, CXCR4-PE-Cy5, CXCR5-APC, and anti–human λ-Ig biotin (BioLegend); and anti–human CD43-PE and anti–human IgD (Miltenyi Biotec).