A nonparenchymal cell fraction from whole liver extracts was isolated upon collagenase and mechanical digestion followed by Percoll (GE Healthcare Life Sciences) gradient centrifugation as previously described [5 (link)]. In parallel, blood samples were collected in EDTA-containing tubes and treated with red blood cell lysis buffer (PharmLyse, BD Biosciences, Germany). Upon removal of red bodies and centrifugation, immune cells were incubated with fluorochrome-conjugated antibodies and characterized according to two different panels, a myeloid panel: CD45-BV510 (103138, BioLegend), 7AAD-PE-Cy5-YG (420404, BioLegend), CD11b-BV711 (101242, BioLegend), F4/80-APC (17-4801-82, eBiosciences), MHC2-Alexa700 (107622, BioLegend), CD11c-PE-Cy7 (25-0114-81, eBiosciences), and Ly6G-FITC (551460, BD Pharmingen) and a lymphoid panel: CD45-BV510 (103138, BioLegend), 7AAD-PE-Cy5-YG (420404, BioLegend), CD3-PE-Cy7 (25-0031-82, eBiosciences), CD4-FITC (11-0041-85, eBiosciences), CD8-PerCpCy5.5 (126610, BioLegend), and NK1.1-BV711 (108745, BioLegend). Labeled cells were then subjected to flow cytometry using a BD Canto II (BD Biosciences) and relative cell numbers were analyzed using FlowJo software (Tree Star).
Ramadori P., Drescher H., Erschfeld S., Fragoulis A., Kensler T.W., Wruck C.J., Cubero F.J., Trautwein C., Streetz K.L, & Kroy D.C. (2017). Genetic Nrf2 Overactivation Inhibits the Deleterious Effects Induced by Hepatocyte-Specific c-met Deletion during the Progression of NASH. Oxidative Medicine and Cellular Longevity, 2017, 3420286.
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