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Preparation of single-cell suspensions of mouse glomeruli for high-throughput analysis

Abstract

The kidney glomerulus is essential for proper kidney function. Until recently, technical challenges associated with glomerular isolation and subsequent dissolution into single cells have limited the detailed characterization of cells in the glomerulus. Previous techniques of kidney dissociation result in low glomerular cell yield, which limits high-throughput analysis. The ability to efficiently purify glomeruli and digest the tissue into single cells is especially important for single-cell characterization methods. Here, we present a detailed and comprehensive technique for the extraction and preparation of mouse glomerular cells, with high yield and viability. The method includes direct renal perfusion of Dynabeads via the renal artery followed by kidney dissociation and isolation of glomeruli by magnet; these steps provide a high number and purity of isolated glomeruli, which are further dissociated into single cells. The balanced representation of podocytes, mesangial and endothelial cells in single-cell suspensions of mouse glomeruli, and the high cell viability observed, confirm the efficiency of our method. With some practice, the procedure can be done in <3 h (excluding equipment setup and data analysis). This protocol provides a valuable technique for advancing future single-cell-based studies of the glomerulus in health, injury and disease.

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Fig. 1: Schematic description of protocol design and steps.
Fig. 2: Glomerulus structure and cell composition.

Data availability

The authors confirm that the data supporting the findings of this study are available within the article, its Supplementary Information and the primary supporting research paper12.

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Acknowledgements

We thank C. Ising, S. Braehler, L. Xie and our Genentech colleagues in the Research Biology, Laboratory Animal Resources, Microscopy and Pathology Departments for their support of this study. This work was supported by Genentech.

Author information

Affiliations

Authors

Contributions

B.K. wrote the manuscript and contributed to the development of the protocol; J-J.C. designed and developed the protocol and revised the manuscript; S.A. contributed to the development of the protocol and writing of the manuscript; and A.S. supervised the experimental design and revised the manuscript.

Corresponding author

Correspondence to Andrey S. Shaw.

Ethics declarations

Competing interests

B.K., S.A. and A.S. are employees of Genentech Research and Early Development. J-J.C. reports employment at Pin Pharmaceuticals.

Additional information

Peer review information Nature Protocols thanks Benjamin Humphreys, Katalin Susztak and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key reference using this protocol

Chung, J.-J. et al. J. Am. Soc. Nephrol. 31, 2341–2354 (2020): https://doi.org/10.1681/ASN.2020020220

Extended data

Extended Data Fig. 1 Preparation of a semi-dulled needle for renal perfusion.

Representative image of a semi-dulled 27½ gauge needle used for direct perfusion via the renal arteries. Images were taken using 1080P Full HD Digital Microscope.

Extended Data Fig. 2 Kidney extraction with renal arteries.

a, A C57BL/6 mouse following the removal of most of the intestine. White arrows indicate right and left kidneys, and the top and bottom of the aorta/inferior vena cava. b, Extraction of the kidney-blood-vessel tissue complex by gently pulling the top part of the aorta/inferior vena cava (left white arrow) while separating attached tissues (lower right white arrow). Dashed white arrow indicates direction of pulling. Appropriate institutional regulatory board permission was obtained for all animal experiments.

Extended Data Fig. 3 Mouse kidney extraction, perfusion and preliminary dissociation.

a, En bloc kidney extraction and transfer to a Petri dish for perfusion. Kidney before (right) and kidney after (left) direct perfusion via the renal artery. b, Kidneys after direct perfusion via the renal arteries. c, Representative image of properly minced kidneys. Images were taken using a 1080P Full HD Digital Microscope. Appropriate institutional regulatory board permission was obtained for all animal experiments performed.

Extended Data Fig. 4 Validation of glomeruli purity following magnetic separation and washes.

a, Representative image of glomeruli and magnetic Dynabeads sample following first enzymatic digestion; before washes, tubular contamination is visible. bd, The sample was washed with 10 ml HBSS−/−, and 10 µl aliquots were taken after the third (b), fourth (c), and fifth (d) washes to check for glomeruli purity under the microscope. e, Representative image of the resuspended glomeruli sample. Images were taken using the Leica Thunder Imaging System. Appropriate institutional regulatory board permission was obtained for all animal experiments.

Extended Data Fig. 5 Confirmation of single-cell suspension purity following magnetic separation and washes.

a, Representative image of a single-cell sample following second enzymatic digestion, before removal of Dynabeads. b, Representative image following removal of magnetic Dynabeads using a magnetic separator. Images were taken using the Leica Thunder Imaging System. Appropriate institutional regulatory board permission was obtained for all animal experiments.

Supplementary information

Supplementary Information

Supplementary Table 1.

Supplementary Video 1

Dissection of aorta and direct renal perfusion. Kidneys are placed in a Petri dish with HBSS−/− and further handled using a dissecting microscope. Dumont forceps are used to remove muscle and fatty tissue over the aorta and inferior vena cava. Then, Vannas spring scissors are used to dissect the abdominal aorta to expose the openings to the renal arteries. Next, the kidney is perfused by inserting the tip of a semi-dulled needle into the opening of the renal artery and slowly injecting the solution. The video was recorded using a Dino-Lite Edge Digital Microscope. Appropriate institutional regulatory board permission was obtained for all animal experiments.

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Korin, B., Chung, JJ., Avraham, S. et al. Preparation of single-cell suspensions of mouse glomeruli for high-throughput analysis. Nat Protoc 16, 4068–4083 (2021). https://doi.org/10.1038/s41596-021-00578-2

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