Isolation and Immunophenotyping of Intrahepatic Lymphocytes

To obtain single-cell suspension, livers were minced by a gentleMACS Dissociator (Miltenyi Biotec, Bergisch Gladbach). Then, red blood cells were lysed (eBioscience™ 1X RBC Lysis Buffer, Thermo Fisher Scientific) and IHLs (intrahepatic lymphocytes) were separated using a 40%/70% Percoll (GE Healthcare) gradient. Subsequently, lymphocytes were stained with anti-CD3, anti-CD4, anti-CD8, anti-Ki-67, anti-B220, anti-Foxp3, and anti-CD62L antibodies. All image acquisitions were performed with an LSRII SORP interfaced with DIVA software (BD Biosciences) as described previously [16 (link), 19 (link)].

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Dywicki J., Buitrago-Molina L.E., Noyan F., Schlue J., Iordanidis K., Manns M.P., Wedemeyer H., Jaeckel E, & Hardtke-Wolenski M. (2022). Splenectomy induces biochemical remission and regeneration in experimental murine autoimmune hepatitis. European Journal of Medical Research, 27, 284.

Publication 2022

gentleMACS Dissociator eBioscience™ 1X RBC Lysis Buffer Percoll LSRII SORP DIVA software

Anti antibodies Anti cd3 Buffer Cd62l Cell Livers Lymphocytes Percoll

Corresponding Organization : University of Duisburg-Essen

Other organizations : Medizinische Hochschule Hannover, University Health Network, University of Toronto

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Variable analysis

independent variables

Mincing of livers using a gentleMACS Dissociator
Lysis of red blood cells using eBioscience™ 1X RBC Lysis Buffer
Separation of intrahepatic lymphocytes (IHLs) using a 40%/70% Percoll gradient

dependent variables

Percentage of lymphocyte subpopulations (CD3+, CD4+, CD8+, B220+, Foxp3+)
Expression of Ki-67 and CD62L on lymphocyte subpopulations

control variables

Staining of lymphocytes with specific antibodies (anti-CD3, anti-CD4, anti-CD8, anti-Ki-67, anti-B220, anti-Foxp3, anti-CD62L)
Image acquisition using an LSRII SORP interfaced with DIVA software (BD Biosciences)

positive controls

Not explicitly mentioned in the provided information.

negative controls

Not explicitly mentioned in the provided information.

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