Biopsy and blister exudate assessments
blisters
LYSIS
Suction blisters and 3 mm biopsy samples were taken from the IMQ-treated areas and control areas at the indicated time points, depending on the cohort and randomization (Blister fluid was collected in a V-bottom plate containing 50 μl 3% sodium citrate (Sigma) in PBS (Gibco) and kept on ice. The plate was centrifuged, and the supernatant was weighed to estimate the volume and then frozen at −80°C for cytokine analysis (Meso Scale Discovery, Rockville, Maryland, USA). The following cytokines were analyzed: TNF, ASC, IL-1β, IL-6, IL-10, IL-8, IFN-γ, and downstream marker for type 1 interferon Mx-A (V-plex proinflammatory panel of MSD and Mx-A protein ELISA kit from BioVendor). The cell pellet was resuspended in RoboSep buffer (StemCell). A cocktail of fluorescent antibodies against cell surface markers was added to the cells and incubated for 30 min on ice. The stained samples were washed with PBS and measured using MACSQuant 16 (Miltenyi Biotec GmbH). Flow cytometry data were analyzed with Flowlogic 7.2 (Inivai). Parallel to the blister fluid, peripheral blood was collected by venipuncture using a sodium heparin vacutainer (BD). Approximately 100 µl of whole blood was treated with red blood cell lysis buffer (eBioscience), washed with PBS, and resuspended in RoboSep buffer. Staining was similar to that of the previously mentioned blister cells and served as a template for the gating strategy. The following antibodies were used: CD56-PE (cat# 130-113-312, Miltenyi Biotec), CD14-PE-Vio615 (cat# 130-110-526, Miltenyi Biotec), CD16-VioBrighT FITC (cat# 130-119-616, Miltenyi Biotec), CD66b-AF700 (cat# 305114, Biolegend), CD19-BV650 (cat# 302238, Biolegend), CD20-BV650 (cat# 302336, Biolegend), HLA DR-APC (cat# 130-111-790, Miltenyi Biotec), CD4-VioBlue (cat# 130-114-534, Miltenyi Biotec), CD8-BV570 (cat# 301038, Biolegend), CD45-VioGreen (cat# 130-110-638, Miltenyi Biotec), CD1c-PE-Vio770 (cat# 130-110-538, Miltenyi Biotec), CD3-APC-Vio770 (cat# 130-113-136, Miltenyi Biotec), 7AAD (cat# 130-111-568, Miltenyi Biotec). An overview of the gating strategy is shown in 