Collection Citation: Dorien De Pooter, Ben De Clerck, Koen Dockx, Domenica De Santis, Sarah Sauviller, Pascale Dehertogh, Matthias Beyens, Isabelle Bergiers, Isabel Nájera, Ellen Van Gulck, Nádia onceição-Neto, Wim Pierson 2024. Robust isolation protocol for mouse leukocytes from blood and liver resident cells for immunology research. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbzpz3gpk/v1
License: This is an open access collection distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this collection and it's working
Created: April 01, 2024
Last Modified: April 12, 2024
Collection Integer ID: 98109
Keywords: Liver, Hepatocytes, NPC, IHL, HBV, Hepatitis, immunology, RNAseq, blood, GentleMACS, liver resident cells for immunology research research, robust isolation protocol for mouse leukocyte, liver resident cell, resident liver cell, viable hepatocyte, protocol for lymphocyte purification, other liver resident cell, important new methodologies for liver, lymphocyte purification, mouse systemic whole blood, higher quality lymphocyte data in downstream analysis, using liver enzymatic digestion, lymphocyte data, efficient removal of red blood cell, protocols for blood, cell rna, liver sinusoidal endothelial cell, mouse leukocyte, liver enzymatic digestion, clean blood sample, hepatitis virus infection, peripheral immune landscape, systemic whole blood, classical mechanical liver disruption method, immunology, liver, sequencing data, optimized purification, immunological response, immunology research research, enzymatic digestion protocol, novel purification method, red blood cell, rna, downstream assay, purification
Abstract
Research on liver-related conditions requires a robust and efficient method to purify viable hepatocytes, lymphocytes and all other liver resident cells, such as Kupffer or liver sinusoidal endothelial cells. Here we describe a novel purification method using liver enzymatic digestion, followed by a downstream optimized purification. Using this enzymatic digestion protocol, the resident liver cells as well as viable hepatocytes could be captured, compared to the classical mechanical liver disruption method. Moreover, single-cell RNA-sequencing demonstrated higher quality lymphocyte data in downstream analyses after the liver enzymatic digestion, allowing for studying of immunological responses or changes. In order to also understand the peripheral immune landscape, a protocol for lymphocyte purification from mouse systemic whole blood was optimized, allowing for efficient removal of red blood cells. The combination of microbeads and mRNA blockers allowed for a clean blood sample, enabling robust single-cell RNA-sequencing data. These two protocols for blood and liver provide important new methodologies for liver-related studies such as NASH, hepatitis virus infections or cancer research but also for immunology where high-quality cells are indispensable for further downstream assays.
Materials
Reagents:
RPMI1640 medium with L-glutamine (Lonza, BE12-702F)
FCS, frozen (0.22µm filtered Gibco by Thermo Fischer Scientific, 011-90005M)
