Af1172

Human skin punch biopsies (Ø 3–5mm) were placed in RPMI 1,640 medium (Gibco) supplemented with 2 mM glutamine (Gibco), 10% heat inactivated fetal calf serum and 100 μg/mL Normocin (InvivoGen). From each donor two (or four) biopsies with identical diameter and thickness were cultured in medium, supplemented with either 10 μg/ml of I/F8-Fc or KSF/Fc (same number of biopsies per condition, to ensure equal average punch sizes for both conditions). Crawled out cells in the culture medium were quantified 24 h later by FACS. For this, biopsies were removed and flow-count fluorospheres (Beckman Coulter) as well as anti-HLA-DR (mouse anti-human HLA-DR FITC; Miltenyi Biotec) and anti-CD86 (mouse anti-human CD86-APC; Miltenyi Biotec) antibodies were added to the medium. In separate experiments, the presence of ALCAM was additionally determined on CD86⁺HLA-DR⁺ DCs that had emigrated from untreated biopsies by co-staining with polyclonal goat anti-ALCAM (R&D Systems, AF1172) or corresponding isotype control (R&D Systems), followed by AlexaFluor594-conjugated secondary antibodies (Invitrogen). Samples were acquired on a Cytoflex S apparatus (Beckman Coulter) using CytExpert software and analyzed with FlowJo software 10.4.0. (Treestar).

Primary antibodies used were: NCAM-1 (1:100, rabbit, Millipore, ab5032; rat, Millipore, mab310); ALCAM (1:50, goat, R&D Systems, AF1172); CNTN2 (1:50, goat, R&D Systems, AF4439); Cx40 (1:500, rabbit, Alpha Diagnostic, Cx40A); PSA (1:500, rabbit, Absolute Antibody, Ab00240-23.0); troponin T (1:100, mouse, Thermo Fisher Scientific MS-295-P0); plakoglobin (1:100, rabbit, Cell Signaling Technology, 2309S). Secondary antibodies used were: donkey anti-rabbit 555 (1:500, Thermo Fisher Scientific, A31572); donkey anti-goat 555 (1:500, Thermo Fisher Scientific, A21432); goat anti-rat 555 (1:500, Thermo Fisher Scientific, A21434); goat anti-rabbit 488 (1:500, Thermo Fisher Scientific, A11034); donkey anti-rabbit 488 (1:500, Thermo Fisher Scientific, A21206); donkey anti-goat 488 (1:500, Thermo Fisher Scientific, A11055); donkey anti-mouse 488 (1:500, Thermo Fisher Scientific, A21202); donkey anti-rat 488 (1:500, Thermo Fisher Scientific, A21208); wheat germ agglutinin (WGA) Alexa Fluor 488 conjugate (1:250, Thermo Fisher Scientific, W11261).