An improved method for isolation and flow cytometric characterization of intrahepatic leukocytes from fatty and fibrotic liver tissues

Abstract

Flow cytometry is an imperative tool to characterize alterations in a wide range of immune cell populations during inflammatory conditions and disease states that affect the liver such as the obesity‐induced non‐alcoholic fatty liver disease and liver fibrosis. Identification and quantification of immune cell subsets from the liver is critically dependent on efficient isolation of intrahepatic leukocytes. The isolation of leukocytes from fatty and fibrotic livers and processing the cells for flow cytometry can be challenging with respect to cell yields, purity and most importantly, the level of autofluorescence resulting from fat deposition. Here, we describe an efficient method for isolating intrahepatic leukocytes from mice fed with high fat diet and propose a strategy to alleviate autofluorescence during phenotyping by multicolor flowcytometry. We also describe a gating strategy for robust identification of granulocytes, pro‐inflammatory, anti‐inflammatory and transitional state monocyte subsets, dendritic cells, B cell, T lymphocyte subpopulations and NK cell subsets. Overall, the procedures described here will allow simultaneous processing of several samples while ensuring reproducible cell isolation and efficient noise reduction required for reliable characterization of intrahepatic leukocytes from the fatty liver tissues.

Profibrogenic role of IL-15 through IL-15 receptor alpha-mediated trans-presentation in the carbon tetrachloride-induced liver fibrosis model

“…Intrahepatic lymphocytes were isolated as described ( 38 ). At sacrifice, liver tissues were collected, rinsed with Krebs–Ringer–Buffer (KRB; 154 mM NaCl, 5.6 mM KCl, 5.5 mM Glucose, 20.1 mM HEPES, 25 mM NaHCO3, pH 7.4) and digested in pre-warmed (37°C) KRB supplemented with 2 mM CaCl 2 , 2 mM MgCl2, 300 CDU (casein digestion units)/mL Collagenase IV (Worthington) and 150 U/mL DNase I (Sigma) using the GentleMACS™ Dissociator (Miltenyi Biotec, Cambridge, MA, US) following the manufacturer’s instructions.…”

“…The supernatant was centrifuged at 300 g for 10 min at 4°C to collect the leukocytes. Cells were further purified using a Percoll gradient as described ( 38 ), rinsed with PEB buffer and used for flow cytometry. Data were acquired on Cytoflex using CytExpert software (BeckMan Coulter, Indianapolis, IN) and analyzed using the FlowJo software (TreeStar Inc, Ashland, OR).…”

Hepatic stellate cell-intrinsic role of SOCS1 in controlling hepatic fibrogenic response and the pro-inflammatory macrophage compartment during liver fibrosis

“…IHLs were isolated by a four steps protocol involving (i) controlled collagenase digestion of minced liver tissue using the GentleMACS tissue dissociator device (Miltenyi Biotech), (ii) microfiltration and sedimentation of hepatocytes and IHLs by differential centrifugation, (iii) Percoll gradient centrifugation of sedimented IHLs to remove fatty debris, and (iv) magnetic selection of hematopoietic cells using anti-CD45 antibody to clarify the resuspended Percoll-sedimented cells as detailed elsewhere ( 38 ). This IHL isolation procedure developed for fatty liver tissues is applicable to fibrotic and normal livers.…”

From anatomy to immunity in the gastrointestinal system

“…The study of local immune responses in the liver is critical to understand these processes but it is frequently hampered by technical difficulties in the isolation and characterization of hepatic immune cells, especially from diseased livers. In this issue, Kandhi et al (2022) describe an improved methodology for the isolation and profiling of intra-hepatic leukocytes, which was validated in animals with experimentally induced NAFLD. Most notably, the new method was very efficient in reducing the intrinsic levels of the samples' autofluorescence, which constitutes a major challenge when studying cells from fatty and fibrotic livers.…”