Multiparametric Flow Cytometry-Based Immunophenotyping of Mouse Liver Immune Cells

Abstract

The liver is a complex organ that governs many types of metabolisms, including energy metabolism and other cellular processes. The liver also plays a crucial role in important functions in immunity, and the activity of liver tissue-associated immunity affects the outcome of many liver pathologies. A thorough characterization of the liver immune microenvironment may contribute to a better understanding of immune signaling, the mechanisms of specific immune responses, and even to improved predictions about therapy outcomes. In this paper, we present an optimized, simple, and rapid protocol to characterize the liver-associated immune cell milieu. We believe that the most suitable technique for obtaining a complex immune cell suspension and for removing contaminating blood cells is to perform mouse liver perfusion, using only phosphate buffer saline. Combining an enzymatic digestion and a mechanical dissociation of liver tissue, followed by cell purification, improves downstream applications. This combination is an essential prerequisite for immune cell determination and characterization. We then demonstrate a flow cytometry-based multiparametric immunophenotyping along with a gating strategy to detect and quantify liver endothelial cells, T cells (helper and cytotoxic), B cells, NK cells, NKT cells, neutrophils, monocytes (subsets included), dendritic cells (subsets included), macrophages and Kupffer cells.

The Biodistribution of Replication-Defective Simian Adenovirus 1 Vector in a Mouse Model

“…17-4801-82, Thermo Fisher Scientific) for 30 min on ice in the dark, washed, resuspended in 0.5 mL PBS containing 1% FBS, and subjected to flow cytometry assay (BD FACSAria II, BD, San Jose, CA, USA). Mouse parenchymal hepatocytes were incubated with a Zombie NIR marker, washed, and similarly assayed with a flow cytometer [ 35 ]. A Zombie NIR marker is an amine-reactive fluorescent dye that is non-permeant to live cells, and therefore, it can help distinguish live and dead cells.…”

Synthetic Stimulator of Interferon Genes (STING) Agonists Induce a Cytokine-Mediated Anti-Hepatitis B Virus Response in Nonparenchymal Liver Cells

“…Mouse liver samples were collected 8–18 weeks post-HDI. Phosphate-buffered saline (PBS)-based liver perfusion was performed in the noninfectious HBV-persistent mouse model (accepted manuscript) as described previously . NPCs were isolated using Liver Dissociation and Debris Removal Kits (Miltenyi Biotec) according to the manufacturer’s protocols.…”

“…Human PBMCs, enriched T cells, and hNPCs/mNPCs were immunophenotyped as previously described ,, (for list of antibodies, see Tables S4 and S5­(hNPCs/mNPCs)). Control samples of unstained cells, positive control for dead cells (cells in PBS incubated for 5 min at 65 °C), fluorescence minus one, and isotype controls were also prepared.…”