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Updated: Jun 29, 2025

Optimizing Isolation and Purification of Murine Glomerular Mesangial Cells

Optimizing Isolation and Purification of Murine Glomerular Mesangial Cells

Published on: March 7, 2025

Optimization of mouse kidney digestion protocols for single-cell applications.

Jake N Robertson¹, Henry Diep¹, Alexander R Pinto^1,2

¹Centre for Cardiovascular Biology and Disease Research, Department of Microbiology, Anatomy Physiology and Pharmacology, School of Agriculture, Biomedicine and Environment, La Trobe University, Bundoora, Victoria, Australia.

Physiological Genomics

|March 25, 2024

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Choosing the right single-cell preparation protocol is crucial for accurate mouse kidney cell analysis. Different protocols significantly alter cellular yields, impacting downstream single-cell RNA sequencing and flow cytometry studies.

Area of Science:

Renal cell biology
Single-cell omics technologies
Biotechnology and bioengineering

Background:

Single-cell technologies enable detailed kidney cell characterization.
Existing protocols for preparing kidney single-cell suspensions vary significantly.
This variability creates disparities in cellular yield and downstream analysis.

Purpose of the Study:

To compare three distinct single-cell preparation protocols for mouse kidney tissue.
To assess the impact of different dissociation protocols on cellular heterogeneity.
To guide researchers in selecting optimal protocols for specific kidney cell enrichment.

Main Methods:

Preparation of single-cell suspensions from male and female C57BL/6 mouse kidneys.
Utilized three protocols: a scRNAseq protocol (P1), Miltenyi Biotec kit (P2), and a lab-developed protocol (P3).

Keywords:

flow cytometry renal single-cell RNA sequencing tissue dissociation

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Flow cytometry was employed to quantify major kidney cell types.

Main Results:

Protocol P2 yielded significantly fewer leukocytes and type B intercalating cells.
Protocols P1 and P3 showed similar yields for most cell types.
Protocol P1 significantly enriched endothelial and myeloid-derived cells; sex differences noted in granulocytes and smooth muscle cells.

Conclusions:

The choice of single-cell digestion protocol critically influences kidney cellular heterogeneity.
Different protocols result in significant variations in cell type yields.
This comparative analysis aids researchers in selecting protocols for targeted kidney cell type enrichment in single-cell studies.