An Optimized Method To Isolate Female Mouse Urethral Epithelium For Single-Cell And Immunofluorescence Analysis

The urethra, lined by epithelial cells, serves as the conduit for urine outflow from the body. The epithelial lining of the urethra comprises distinct cell types with gene signatures indicative of roles in antimicrobial and immune defense. Resident macrophages embedded in the urethral epithelial layer represent a transcriptionally distinct subtype with purported roles in immune surveillance and antigen presentation. Urinary pathogens ascend up the urethra to reach the bladder. A study of the urethral epithelial lining and associated immune cells will shed light on host defense mechanisms in the lower urinary tract. A central goal of this protocol is to provide optimized methods for dissecting the female mouse urethra and isolating the epithelial lining for downstream analyses. The protocol describes an optimized method for isolating epithelial tissue from the female mouse urethra by gentle enzymatic and mechanical separation. Subsequent to epithelial isolation, methods for gentle enzymatic digestion of isolated urethral epithelium and associated immune cells to obtain high-viability single-cell suspensions are described. The protocol also details methods for flow cytometry analysis of the isolated mouse urethral epithelial cells and epithelial-associated immune cells. Additionally, methods to generate stratified 3D urethral epithelial organoids from isolated epithelial cells are presented here. The protocol also details an optimized method for whole-mount immunostaining of urethral epithelial sheets, which can be used to observe the morphology and molecular structures of mouse urethral epithelium and associated immune cells. Overall, the methods described here for isolation and single-cell digestion of mouse urethral epithelial cells enable downstream analyses, including immunostaining, flow cytometry, organoid generation, and single-cell RNA-sequencing.