Computational protocol: Absence of Tumor Necrosis Factor Supports Alternative Activation of Macrophages in the Liver after Infection with Leishmania major

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Protocol publication

[…] Multicolor flow cytometry was performed following an established protocol (). Cells were stained first for surface marker expression with rat antimouse CD45 (Biotinylated; 30-F11; BD Biosciences), rat antimouse Ly6C (FITC; clone HK 1.4; BioLegend, WA, Australia), rat antimouse F4/80 (APC-Cy7; clone BM8; eBioscience, VIC, Australia), and rat antimouse CD11b (PerCP-Cy5.5; clone M1/70; BD Biosciences). For intracellular flow cytometry the cells were fixed with FOXP3 Fix/Perm buffer and permeabilized with FOXP3 Perm buffer (BioLegend) according to the manufacturer’s protocol. Intracellular proteins were targeted with rat antimouse CD206 (PE; clone C068C2; BioLegend), rat antimouse IL-6 (PE; clone MP5-20F3; BD Biosciences), rat antimouse IFN-γ (PE; clone XMGI-2; BD Biosciences), rabbit anti-L. major [clone V121 ()], and mouse antimouse Arg-1 (PE; polyclonal antiserum; R&D Systems, Sydney, NSW, Australia). Streptavidin conjugated to V500 (BD Biosciences) was used to reveal biotinylated primary mAbs. Cells were acquired on a BD FACSCanto II flow cytometer using BD FACSDiva version 6.1.3 (BD Biosciences) and analyzed with FlowJo software version 10.1(Tree Star Inc., Ashland, OR, USA). For flow cytometric cell sorting, two populations defined by CD45+F4/80+CD11b+Ly6Clow and CD45+F4/80+CD11b+Ly6Chi were sorted using a Beckman Coulter Astrios MoFlo. For liver DC marker comparison, CD11c (PE-Cy7; clone HL3; BD Biosciences) was used. Bone marrow-derived cells were stained with CD11b (FITC; clone M1/70; BD Biosciences), CD11c (PE-Cy7; clone HL3; BD Biosciences), F4/80 (APC-Cy7; clone BM8; eBioscience), CD206 (PE; clone C068C2; BioLegend), and M-CSFR (APC; clone AFS98; Biolegend) as experiments required. […]

Pipeline specifications

Software tools BD FACSDiva, FlowJo
Application Flow cytometry
Organisms Leishmania major, Mus musculus
Diseases Neoplasms