Flow cytometry analyses

For surface staining, 2 × 10⁶ skin cells or LN cells were first incubated with anti-CD16/CD32 antibody [0.5:25 (volume of antibody: volume of staining FACS buffer); clone 93, eBioscience] to block unspecific binding, followed by surface staining with the following fluorochrome-conjugated antibodies in FACS buffer: CD45 APC-eFluor780 (0.06:25, clone 30-F11), CD45R/B220 APC (1.2:25, clone RA3-6B2), GL7 PE (1.25:25, clone GL-7), Gr-1 PE (Ly-6G/Ly-6C) (0.02:25, clone RB6-8C5), CD8a PerCP-Cy5.5 (0.5:25, clone 53-6.7), TCRβ PerCP-Cy5.5 (1:25, clone H57-597), CD3 FITC (1:25, clone 145-2C11), Ly-6C PE-Cy7 (0.3:25, clone HK1.4), Ly-6G APC (1:25, clone 1A8-Ly6g), CD49b biotin (0.5:25, clone DX5) and streptavidin APC (0.5:25) were from eBioscience; Gr-1 FITC (0.05:25, Ly-6G/Ly-6C) (clone RB6-8C5), Siglec-F PE (0.5:25, clone E50-2440), CD95 PE-Cy7 (1:25, clone Jo2), CD19 FITC (1:25, clone 1D3), CXCR5 biotin (1.5:25, clone 2G8), IgE biotin (0.5:25, clone R35-72) and streptavidin BV605 (0.5:25) were from BD Biosciences; TCRβ PE-Cy7 (0.5:25, clone H57-597), CD4 BV421 (0.5:25, clone GK1.5), PD-1 PE-Cy7 (2:25, clone RMP1-30), CD45R/B220 PE-Cy7 (1.2:25, clone RA3-6B2), IgG1 PerCP-Cy5.5 (1:25, clone RMG1-1) were from Biolegend.

For IL-1β intracellular staining, cells were first stained for surface markers and then stained for IL-1β using Fixation/Permeabilization Kit (BD Biosciences, Cat No. 554715). Briefly, cells were fixed and permeabilized with Fixation/Permeabilization solution for 20 min. After wash and centrifugation, cells were resuspended in Perm/Wash buffer containing anti-IL-1β PE antibody (2:100, clone 166931, R&D Systems) for 30 min. Cells were washed and resuspended in FACS buffer for analyses.

To eliminate dead cells, propidium iodide was used for surface staining, and Fixable Viability Dye eFluor 506 (0.1:100, eBioscience Cat. 65-0866-18) was used for intracellular staining. Samples were passed on LSRFortessa X-20 (BD) and data were collected with BD FACS DIVA v8 and analysed with FlowJo.