Protocols for endothelial cell isolation from mouse tissues: kidney, spleen, and testis

Abstract

Endothelial cells (ECs) exhibit phenotypic and functional tissue specificities, critical for studies in the vascular field and beyond. Thus, tissue-specific methods for isolation of highly purified ECs are necessary. Kidney, spleen, and testis ECs are relevant players in health and diseases such as chronic kidney disease, acute kidney injury, myelofibrosis, and cancer. Here, we provide tailored protocols for rapid and reproducible EC purification established for scRNA sequencing from these adult murine tissues using the combination of magnetic- and fluorescence-activated cell sorting. For complete details on the use and execution of these protocols, please refer to Kalucka et al. (2020) and Dumas et al. (2020).

Keywords: Cell isolation; Flow Cytometry/Mass Cytometry; Single Cell.

Graphical abstract

Figure 1

EC isolation from mouse kidney and glomeruli (A) Detailed scheme illustrating isolation of ECs from kidney. (B and C) Representative FACS plots for gating strategy to sort ECs from kidney glomeruli (B) based on sorting live/CD45^-/CD73^-/CD102⁺ cells and from renal parenchyma (C) based on sorting live/CD45^-/CD31⁺ cells.

Figure 2

EC isolation from mouse spleen (A) Detailed scheme illustrating isolation of ECs from spleen. (B) Representative FACS plots for gating strategy to sort ECs from spleen based on sorting live/CD45^-/CD11b^-/CD31⁺ cells.

Figure 3

EC isolation from mouse testis (A) Detailed scheme illustrating isolation of ECs from testis. (B) Representative FACS plots for gating strategy to sort ECs from testis based on sorting live/CD45^-/CD31⁺ cells.

Figure 4

Characterization of the renal and glomerular EC sample purity (A) t-SNE (t-distributed stochastic neighbor embedding) visualization of scRNA-seq analyses on ECs isolated from mouse renal parenchyma showing representative EC gene markers expression. Red arrowheads are pointing at cells highly expressing the marker gene. Color scale: red, high expression; blue, low expression. (B) Top: t-SNE visualization of renal parenchyma (non-) ECs color coded per cell type. Red arrowheads are pointing at non-ECs. Dotted line circles the fraction of glomerular ECs. Bottom: bar plot illustrating the quantification of EC and non-ECs. (C) t-SNE visualization scRNA-seq analyses on ECs isolated from mouse glomeruli showing representative EC and non-EC gene markers expression and the number of genes expressed per cells. Red arrowheads are pointing at cells highly expressing the marker gene (or cells with low gene number expression). Color scale: red, high expression; blue, low expression. (D) Top: t-SNE visualization of glomerular (non-) ECs color coded per cell type. Red arrowheads are pointing at non-ECs. Bottom: bar plot illustrating the quantification of EC and non-ECs.

Figure 5

Characterization of the splenic and testicular EC sample purity (A) t-SNE visualization scRNA-seq analyses on ECs isolated from mouse spleen showing representative EC gene and non-EC markers expression. Red arrowheads are pointing at cells highly expressing the marker gene. Color scale: red, high expression; blue, low expression. (B) Top: t-SNE visualization of spleen (non-) ECs color coded per cell type. Red arrowheads are pointing at non-ECs. Bottom: bar plot illustrating the quantification of EC and non-ECs. (C) t-SNE visualization scRNA-seq analyses on ECs isolated from mouse testis showing representative EC markers expression and the number of genes expressed per cells. Red arrowheads are pointing at cells highly expressing the marker gene (or cells with low gene number expression). Color scale: red, high expression; blue, low expression. (D) Top: t-SNE visualization of testis (non-) ECs color coded per cell type. Red arrowheads are pointing at contaminating cells. Bottom: bar plot illustrating the quantification of EC and contaminating cells.