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.