Anti tnfα alexafluor647

PBMCs were thawed and cultured overnight as described above prior to stimulation with either IL-12 (0.5 ng/ml) or IL-15 (20 ng/ml, both purchased from BioLegend, San Diego, CA, USA) with or without K562 cells (PBMC:K562 ratio 2:1) as described (11 (link)). Cells were then washed and stained with anti-human lineage cocktail 3 and anti-CD56-PE as explained above. Cells were washed again, fixed, and permeabilized with the Cytofix/Cytoperm Kit (BD Biosciences, San Jose, CA, USA) and stained with anti-IFN-γ-PerCPCy5.5 and anti-TNFα-AlexaFluor647.

The maturation of DCs was examined by staining with fluorescence‐conjugated antibodies including anti‐CD11c‐FITC (eBioscience, catalog: 11‐0114‐85), anti‐CD86‐APC (eBioscience, catalog: 17‐0862‐82), and anti‐MHC Class II‐PE (eBioscience, catalog: 12‐5321‐82). To analyze the T‐cell subsets in spleens and draining lymph nodes of the immunized mice, single cell suspensions prepared from these samples were examined by flow cytometry. The following primary antibodies were used: anti‐CD3e‐PerCP‐Cyanine5.5 (eBioscience, catalog: 45‐0031‐82), anti‐CD8a‐PE (eBioscience, catalog: 12‐0081‐82), anti‐CD4‐FITC (eBioscience, catalog: 11‐0041‐85), anti‐CD25‐APC (eBioscience, catalog: 17‐0251‐82), anti‐Foxp3‐PE (eBioscience, catalog: 12‐4771‐82), anti‐IFN‐γ‐FITC (BD Bioscience, catalog: 554 411), anti‐TNF‐α‐Alexa Fluor 647 (BD Bioscience, catalog: 557 730), and anti‐CD16/CD32 (eBioscience, catalog: MA5‐18012). Flow data were acquired on a CytoFLEX and analyzed using CytExpert (Beckman Coulter, USA) and FlowJo (TreeStar, USA) softwares.