Hepatic Immune Cell Profiling

Single cell suspensions were derived from hepatic digestion as described and stained with directly conjugated monoclonal antibodies or isotype controls (all eBioscience) [3 (link), 17 (link)]. To determine IL-17A and IL-17F production, total single cells were stimulated for 5 h with 50 ng/ml PMA (Sigma-Aldrich, St. Louis, MO) and 1 μg/ml Ionomycin (Calbiochem), in presence of brefeldin A (10 μg/mL, Sigma-Aldrich). Subsequently, flow cytometry was used to enumerate immune cell populations as described [18 (link)–20 (link)]. Briefly, cells were treated with FACS fix buffer (BD Biosciences) for 15 minutes, washed and incubated in PBS supplemented with 2% FBS and were subsequently stained with Live/Dead stain (Zombie UV Dye: Biolegend) and with directly-conjugated monoclonal antibodies to CD45-AF700 (104), TCRβ-PE (H57-597), CD4-APCef780 (GK1.5), CD8-ef450 (53–6.7), CD11b-PE (M1/70), CD11c-Percp (N418), F4/80-APCef780 (BM8), Ly6C-Percp (HK1.4) Gr1-FITC (RB6-8C5), NK1.1-PECy7 (PK136), B220-ef450 (RA3-6B2), IL-17RA-APC (PAJ-17R), TNF-BV650 (MP6-XT22), IL-17A-Percp (17B7) and IL-17F-PE (18F10) [all antibodies from e-Bioscience] for 30 minutes. Flow cytometry data were then collected using a LSR Fortessa (BD) flow cytometer and analyzed using FlowJo X software (vX0.7).

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Giles D.A., Moreno-Fernandez M.E., Stankiewicz T.E., Cappelletti M., Huppert S.S., Iwakura Y., Dong C., Shanmukhappa S.K, & Divanovic S. (2016). Regulation of Inflammation by IL-17A and IL-17F Modulates Non-Alcoholic Fatty Liver Disease Pathogenesis. PLoS ONE, 11(2), e0149783.

Publication 2016

isotype controls PMA brefeldin A Live/Dead stain (Zombie UV Dye: MP6-XT22 IL-17A-Percp (17B7) IL-17F-PE (18F10) LSR Fortessa

Antibodies Brefeldin a Buffer Cd11b Cells Digestion Fitc Flow cytometry Il 17a Il 17f Il 17ra Ionomycin Isotype Monoclonal antibodies Populations Stain Tcrβ

Corresponding Organization : Cincinnati Children's Hospital Medical Center

Other organizations : Tokyo University of Science, The University of Texas MD Anderson Cancer Center

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Variable analysis

independent variables

PMA (50 ng/ml)
Ionomycin (1 μg/ml)

dependent variables

IL-17A production
IL-17F production
Immune cell populations

control variables

Brefeldin A (10 μg/mL)
Directly conjugated monoclonal antibodies
Isotype controls
FACS fix buffer
Zombie UV Dye (Live/Dead stain)
Directly-conjugated monoclonal antibodies (CD45, TCRβ, CD4, CD8, CD11b, CD11c, F4/80, Ly6C, Gr1, NK1.1, B220, IL-17RA, TNF, IL-17A, IL-17F)

positive controls

Not explicitly mentioned

negative controls

Isotype controls

Annotations

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