Fixation permeabilization wash buffer

For characterization of in vitro-cultured DCs, CC tissue-isolated cells and ex vivo-stimulated cells, cells were blocked with Fc receptor (Human TruStain FcX™, 422302, Biolegend), and incubated for 30 min in the dark with antibodies recognizing specific surface proteins. An Annexin V apoptosis detection kit with propidium iodide (PI; BioLegend, 640932) was used to analyze cell apoptosis. For the detection of intracellular proteins, cells were stimulated for 6 hours with 10 μg/ml brefeldin A and 2 μM ionomycin (Absin) before staining. Cells were then fixed and permeabilized using fixation/permeabilization wash buffer (BioLegend) and stained with antibodies for 30 min in the dark. For Ki67 staining, cells were incubated in 70% ethanol for 1 hour at −20°C and then stained with an anti-Ki67 antibody (350519; BioLegend). All antibodies used in this study are listed in Supplementary Table 1. All samples were run on a CytoFLEX platform (Beckman Coulter, Brea, CA, USA) and analyzed using FlowJo version 10.8 software (BD Biosciences).