Single cell suspensions were derived from hepatic homogenate and stained with directly conjugated monoclonal antibodies or isotype controls. Staining for cytokine expression was completed after 4 hours of stimulation with 50 ng/mL Phorbol 12-Myristate 13-acetate (PMA; Promega), 1 μg/mL Ionomycin (Calbiochem) and 10μg/mL Brefeldin A (Gold Bio). Data collection and analysis were done as previously described11 (link),63 (link). Briefly, cells were stained with Live/Dead stain (Zombie UV Dye: Biolegend) and with directly-conjugated monoclonal antibodies to CD45-AF700 (104), CD11b-PE (M1/70), F4/80-APCef780 (BM8), Ly6C-Percp (HK1.4), Gr1-FITC (RB6-8C5), NK1.1-PECy7 (PK136), TCRβ-PE (H57-597), CD4-APCef780 (GK1.5), CD8-ef450 (53-6.7), TNF-BV650 (MP6-XT22), IL-17A-Percp (17B7) and IL-17F-PE (18F10) [all antibodies from e-Bioscience]. Flow cytometry data were then collected using a LSR Fortessa (BD) flow cytometer and analyzed using FlowJo X software (vX0.7).
Protocol detail
Cytokine Expression Profiling of Hepatic Immune Cells
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Brefeldin A
Zombie UV Dye
F4/80-APCef780
Ly6C-Percp
Gr1-FITC
TCRβ-PE
CD4-APCef780
CD8-ef450
TNF-BV650 (MP6-XT22),
IL-17A-Percp (17B7)
IL-17F-PE (18F10)
LSR Fortessa
Antibodies
Brefeldin a
Cd11b
Cells
Cytokine
Fitc
Flow cytometry
Gold
Il 17a
Il 17f
Ionomycin
Isotype
Monoclonal antibodies
Promega
Stain
Tcrβ
Corresponding Organization :
Other organizations : University of Cincinnati Medical Center, Cincinnati Children's Hospital Medical Center, University of Lübeck, University of Cincinnati, University Hospital Schleswig-Holstein, Joslin Diabetes Center, Heinrich Heine University Düsseldorf, Deutsches Diabetes-Zentrum e.V., Helmholtz Zentrum München, German Center for Diabetes Research, Tokyo University of Science, Bill & Melinda Gates Foundation
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Variable analysis
independent variables
- Phorbol 12-Myristate 13-acetate (PMA; Promega)
- Ionomycin (Calbiochem)
- Brefeldin A (Gold Bio)
- Cytokine expression
- Cell populations (CD45, CD11b, F4/80, Ly6C, Gr1, NK1.1, TCRβ, CD4, CD8)
- Intracellular cytokines (TNF, IL-17A, IL-17F)
- Cell types (single cell suspensions derived from hepatic homogenate)
- Staining with isotype controls
- Stimulation with 50 ng/mL Phorbol 12-Myristate 13-acetate (PMA; Promega), 1 μg/mL Ionomycin (Calbiochem) and 10μg/mL Brefeldin A (Gold Bio) for 4 hours
- Staining with isotype controls
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