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

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.
Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: D.DP, B.DC, S.S, P.D, M.B, I.B, I.N, E.VG, N.CN and W.P were employed by Janssen Research and Development at the time of the research and may be Johnson and Johnson stockholders. All other authors declare no conflicts of interest. This does not alter our adherence to the PLOS ONE policies on sharing data and materials, restrictions do apply to the sharing of any further data on the CAM-A compound and to the sharing of CAM material.
(Copyright: © 2024 De Pooter et al. This is an open access article 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.)