# Robust isolation protocol for mouse leukocytes from blood and liver resident cells for immunology research

## TL;DR

This paper introduces new methods to isolate liver and blood immune cells in mice, improving the quality of data for studying diseases like hepatitis and cancer.

## Contribution

A novel enzymatic digestion and purification protocol for liver and blood cells that enhances cell viability and RNA-sequencing data quality.

## Key findings

- Enzymatic digestion captures viable liver resident cells and hepatocytes better than mechanical disruption.
- Optimized protocols for blood lymphocytes improve single-cell RNA-sequencing data quality.
- Combined methods support high-quality immunological and liver disease research.

## 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.