Computational protocol: Specific roles for dendritic cell subsets during initiation and progression of psoriasis

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Protocol publication

[…] Single cell suspensions were stained with fluorescent antibodies for 30 min on ice after blocking Fc-receptors with anti-CD16/CD32 antibody. For intracellular IL-17 staining of DKO* skin, dermal and epidermal cell suspensions were pooled and stimulated for 4.5 h with 500 ng/ml PMA (Sigma) and 500 ng/ml ionomycin (Sigma) in the presence of GolgiPlug (BD Biosciences) for the last 4 h.For a list of monoclonal antibodies used, see Supplementary Table S1. Gating for flow cytometric analysis in Fig B was performed as previously described (Tamoutounour et al, ). In brief, subsets were gated as:CD11b+DCs (CD11b+CD64−CCR2+Ly-6C−MHC-II+ CD24lo)MHC-IIlomoDCs (CD11b+CD64loCCR2+Ly-6ChiMHC-IIlo CD24−)MHC-II+moDCs (CD11b+CD64loCCR2+Ly-6CloMHC-II+ CD24−)MHC-II+ dermal macrophages (CD11b+CD64hiCCR2loLy-6CloMHC-II+ CD24−)Dead cells were excluded by fixable dead cell stainings (Fisher Scientific, ebioscience). For intracellular stainings, cells were fixed in 2% PFA (Roth) and subsequently permeabilized using PermWash buffer (BD Biosciences). For Ki67, IL-17, and FoxP3 stainings, a FoxP3 Fix/Perm buffer set (Biolegend) was used. Flow cytometry was performed on a LSRFortessa cell analyzer (BD Biosciences), and data were analyzed with FlowJo 7.6.4 software (Treestar). All flow cytometric gatings were performed on live cells following exclusion of doublets with FSC-A/FSC-H. Gates for activation markers and intracellular FoxP3, BrdU, Ki-67, and IL-17 stainings were set according to a corresponding isotype control. Numbers in flow cytometric plots and within graphs depicting quantifications of flow cytometric stainings indicate the percentage of a population of live single cells. [...] Experiments were performed at least two times, and data are represented as mean ± standard error of the mean (SEM). All graphs and statistical analyses were generated GraphPad Prism4 and Adobe illustrator software. Unpaired two-tailed student's t-test, Mann–Whitney U-test, and Wilcoxon signed-rank test were used to assess statistical significance (*P < 0.05, **P < 0.01, ***P < 0.001), as indicated in the figure legends. […]

Pipeline specifications

Software tools FlowJo, Adobe Illustrator
Applications Miscellaneous, Flow cytometry
Organisms Mus musculus, Homo sapiens
Diseases Brain Diseases, Psoriasis, Arthritis, Psoriatic