Liver Immune Cell Subset Profiling

Characterization of immune cell subsets in the liver was performed essentially as described previously^{21 (link)}. The individual samples were analyzed with a LSRII/Fortessa flow cytometer (BD Biosciences) and the FlowJo software Vx (Treestar). All indicated antibodies and reagents were titrated to determine optimal concentrations. CompBeads (BD) were used for single-color compensation to create multi-color compensation matrices. For gating, fluorescence minus one controls were used. The instrument calibration was controlled daily using cytometer setup and tracking beads (BD). Single cell suspensions were created using the Miltenyi Liver Dissociation Kit (No. 130-105-807) and the GentleMACS isolator (Miltenyi) using standard protocols. The following antibodies were used: anti-CD3-PE-CF594, anti-CD4-V500, anti-CD11c-AlexaFluor700, anti-CD19-APC-H7, anti-CD326 (EPCAM)-BV711, anti-Ly6C-PerCP-Cy5.5 (all from BD), anti-CD8-eFluor650, anti-CD11b-eFluor605NC (eBioscience), anti-CD45-VioBlue, anti-CD49b-PE, anti-MHC-II-APC (Miltenyi), anti-F4/80-PE-Cy7, anti-Ly6G-APC-Cy7 (Biolegend). A gating strategy is provided in the supplementary material and methods (Fig. S1).

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Waidmann O., Pleli T., Weigert A., Imelmann E., Kakoschky B., Schmithals C., Döring C., Frank M., Longerich T., Köberle V., Hansmann M.L., Brüne B., Zeuzem S., Piiper A, & Dikic I. (2020). Tax1BP1 limits hepatic inflammation and reduces experimental hepatocarcinogenesis. Scientific Reports, 10, 16264.

Publication 2020

LSRII/Fortessa CompBeads GentleMACS isolator anti-CD3-PE-CF594 anti-CD4-V500 anti-CD11c-AlexaFluor700 anti-CD19-APC-H7 anti-Ly6C-PerCP-Cy5.5 anti-CD45-VioBlue anti-CD49b-PE anti-MHC-II-APC anti-F4/80-PE-Cy7 anti-Ly6G-APC-Cy7

Anti cd3 Antibodies Cd11b Cell Cy5 5 Epcam Fluorescence Liver

Corresponding Organization : University Hospital Frankfurt

Other organizations : University Hospital Heidelberg, Heidelberg University

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Variable analysis

independent variables

Dissociation of liver tissue using Miltenyi Liver Dissociation Kit and GentleMACS isolator

dependent variables

Characterization of immune cell subsets in the liver

control variables

Calibration of instrument using cytometer setup and tracking beads (BD) controlled daily
Titration of antibodies and reagents to determine optimal concentrations
Use of fluorescence minus one controls for gating

controls

Positive control: CompBeads (BD) used for single-color compensation to create multi-color compensation matrices
Negative control: Not explicitly mentioned

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