Name: An Optimized Method To Isolate Female Mouse Urethral Epithelium For Single-Cell And Immunofluorescence Analysis (Video) | JoVE Journal
Uploaded: 2026-06-12T14:30:00.000+00:00
Duration: 11 min 2 s
Description: Learn an optimized method to isolate female mouse urethral epithelium for single-cell and immunofluorescence analysis. This protocol enables flow cytometry, organoid generation, and studies of host defense mechanisms in the lower urinary tract.

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5 minutes

We acknowledge the Animal Care and Resource Centre at the National Centre for Biological Sciences (NCBS), Bangalore Life Sciences Cluster (BLiSC) for their services. We acknowledge Yousuf Khan and the Communications Office at BRIC-inStem for photography. We thank Dr. Sudarshan Gadadhar for access to AxioObserver microscope and Apotome. This work was supported by DBT/Wellcome Trust India Alliance Early Career Fellowship (Ref: IA/E/21/1/506274) to D.B.J. and Department of Biotechnology (DBT), Government of India Institutional core funds to inStem.

The authors have no competing interests. No AI tools were used in this article.

Epithelial barriers in the body are critically important for host defense. These are specialized compartments with multiple epithelial cell types and associated resident immune cells. Epithelial barriers change over developmental time, acquiring host defense gene expression often in response to microbiota or environmental exposures. Defects to epithelial integrity or gene expression may influence host defense15. It is essential to study epithelial tissue in isolation to understand transcriptomic signatures as well as to investigate cell-cell contacts and epithelial integrity. Currently no methods have been described in detail that are optimized for the isolation of urethral epithelial tissue from the female mouse. While the bladder epithelium can be isolated by only mechanical separation16, the urethral epithelium is present inside the stromal tube of the urethra and requires a combination of enzymatic and mechanical separation to isolate epithelial tissue.
Here, the authors present an optimized method for isolation of epithelial sheets from the urethral lumen of female mice. The method can be used for generating high viability single-cell suspensions of urethral epithelial cells for downstream applications like flow cytometry, organoid culture and immunostaining. The compatibility of this method with single-cell RNA-sequencing workflows was previously demonstrated by the same group5. With modifications and further optimization, the protocol presented here can be adapted to extract epithelial tissues from other tubular organs for downstream applications.
The key steps in the protocol involve careful dissection of the full length adult female mouse urethra, enabling the study of regional differences along the proximal-distal axis. Following this, the protocol uses gentle enzymatic digestion to loosen the epithelial&#160;lining of the urethra from the surrounding stroma. The enzymatic digestion loosens the epithelial to allow mechanical separation by gently squeezing the urethral tube between forceps to the release the epithelial tissue. The researchers have carefully optimized the timing for trypsin digestion to enable isolation of urethral epithelium with high viability (&#62; 80%) as sheets for immunostaining or for downstream single-cell isolation (Figure 3, Supplementary File 1). Single-cell digestion of urethral epithelium revealed 80&#8211;90% of epithelial cells in urethral epithelial isolates. From a previous study, authors identified that non-epithelial cells in this preparation are mostly comprised of epithelial-associated immune cells and vascular endothelium5. The current method for single-cell isolation yields high quality single-cell suspensions for downstream applications like flow cytometry and single-cell RNA-sequencing by eliminating cell clumps through mechanical disruption via small gauge needle aspiration and removal of RBCs by gentle lysis.
The results demonstrate that this is method can be used to enrich compartment specific immune cells (Figure 3). Epithelium isolated from the proximal and distal urethra can also be studied separately. Additionally, the authors demonstrated that isolated epithelial cells can be used for the generation of urethral organoids which have&#160;not been described previously (Figure 4). These results also demonstrate regional variability in organoid generation capacity in the female urethra. While the author&#8217;s recent studies have shown proximal-distal differences in epithelial gene expression and immune cell organization in the female mouse urethra5, the current study shows that proximal urethral epithelial cells form fewer organoids than epithelium isolated from the distal urethra (Figure 4). One limitation of this spatial demarcation into proximal and distal urethra is that there is no anatomical landmark that can be used at the time of dissection to distinguish between these two areas. In this study, the authors have divided the length of the urethra by half to obtain proximal and distal compartments. The epithelial isolation method described here can be used to further characterize the differences in gene expression and cellular composition between the proximal and distal urethral epithelium.
While the optimized protocol presented here is accessible and reproducible, some steps require practice and careful calibration. In particular, the dissection method shown here requires careful extraction of the urethral tube from below the pubic symphysis while preserving the entire length of the urethra. Additionally, care must be taken to remove any vaginal tissue attached to the dorsal urethral wall after dissection. Fat present around the bladder neck should be carefully removed to reduce adipocyte&#160;contamination during downstream applications like single-cell RNA-sequencing. Further, trypsin digestion should be carefully optimized with each batch of enzyme to ensure consistent digestion. Over-digestion of tissue may lead to loss of cell viability. The authors have carefully optimized the time for trypsin digestion, indicating a 15&#8211;30 min time window for the incubation steps at 4 &#176;C and 37 &#176;C. This can be further optimized based on the batch of enzyme used. It is also critical to mince tissues into small pieces for efficient single-cell digestion. The authors have used Accutase for further single-cell digestion as it results in a gentle dissociation. In case of a reduction in cell viability, the time for Accutase incubation can be reduced. Additionally, the authors have included Rock inhibitor in the Accutase digestion mix to enhance cell survival. DNase digestion is recommended to reduce cell clumping by digesting extracellular DNA released from dead and dying cells. The authors also recommend the use of RBC lysis buffer to reduce red blood cell contamination that would confound downstream results like flow cytometry and single-cell RNA-sequencing.
One limitation of the epithelial isolation method presented here is that it is has been optimized for the female mouse urethra. The male mouse urethra is more anatomically complex, with changing epithelial architecture from the proximal to the distal urethra. Additionally, the method can only isolate the epithelial cells lining the urethral lumen. Urethral secretory glands connected to the urethral lumen via short epithelial ducts cannot be isolated as these are embedded in stromal tissue. Instead, these epithelial structures are enriched in the stromal compartment remaining after isolating the urethral epithelial lining (Figure 3).
Despite these limitations, this method has enabled important insights into urethral epithelial architecture and will allow further investigation into epithelial-associated immune cells via visualization of epithelial-associated macrophages in situ. The method has already been demonstrated to be compatible with single-cell RNA-sequencing workflows to identify cellular heterogeneity in the urethral epithelium and associated immune cells5. Additionally, the capability to isolate epithelium from the proximal and distal urethra will enable studies to understand spatial differences in the urethra. Significantly, the epithelial cell digestion method yields viable single cells that can generate urethral epithelial organoids that will be useful to study host-pathogen interactions and epithelial biology of the urethra. Overall, these methods add to the tool kit to enable deeper investigations into urethral epithelial biology.

The mouse lower urinary tract (LUT) consists of two parts: the bladder and the urethra. During embryonic stages, the structures of the LUT develop from the cloaca, a transient endoderm-derived structure located at the caudal hindgut. The ventral portion of the cloaca, the urogenital sinus, forms the bladder anteriorly and the urethra posteriorly1. The lining of the bladder is well studied. It forms a tight barrier against urine components through apical crystalline plaques comprised of uroplakin proteins2,3,4. While the bladder lining is a transitional epithelium, the urethra shows differences in epithelial architecture from the bladder neck to the distal urethral opening. Additionally, the urethra shows sexual dimorphism in epithelial structures and cellular architecture. The male mouse urethra is comprised of transitional epithelium at the bladder neck, followed by columnar and stratified epithelium. The female mouse urethra is lined by transitional epithelium at the bladder neck and multi-layered stratified non-squamous epithelium dotted by small ducts ending in secretory glands towards the distal end5.
Females are more susceptible to urinary tract infections (UTIs), predominantly caused by uropathogenic bacteria that ascend from the gut through the urethra to infect the bladder6. The urethra is the first surface in the LUT that comes in contact with ascending urinary pathogens. The function of urethral epithelial cells, especially their role as an active immunological barrier, has been understudied. Recent work from the same group provided a single-cell resolution spatial map of the adult female mouse urethra5. It identified a triple-layered urethral lining with basal, intermediate, and apical cells. Interspersed in the epithelial lining are rare neuroendocrine cells5,7. Towards the middle of the urethra, small ducts branch from the main trunk of the urethra, ending in clusters of secretory glands, embedded in the stromal compartment of the urethra. Transcriptomic data revealed that the epithelial cells in the urethra express immunomodulatory and antimicrobial genes that may shape host defense5. Studying the properties and attributes of urethral epithelial cells related to their defense potential and immunomodulatory functions may provide novel strategies to combat UTIs.
Using the mouse to study the urethral epithelium allows temporal studies and investigation of sexual dimorphism. Given the similarities of the mouse to the human in terms of cellular architecture of the bladder and urethra1, the mouse has been extensively used to study embryonic development of the lower urinary tract1,8,9. Mice have also been used in transurethral instillation models to study UTI caused by urinary pathogens10,11,12. Lower urinary tract tissue is difficult to access in patients unless undergoing resection for cancer or other disorders. A source of consistent, healthy human tissue is difficult to access and the mouse provides a useful model to investigate tissues in the lower urinary tract for studies on tissue homeostasis, repair and response to inflammatory insults. The female mouse and human urethral lining are both comprised of transitional epithelium at the bladder neck and stratified non-squamous, non-keratinized epithelium towards the distal end. Squamous epithelium is observed at the distal end of the human female urethra, although variability is observed in human female urethral lining with age5,13. Although comparisons of the urethral lining at the transcriptome level have not been carried out between the human and mouse urethra, the mouse represents a useful model to capture epithelial patterning, epithelial-immune interactions, and response to infections in the urethra.
In this study, the authors describe methods for dissection of the full-length adult female mouse urethra, isolation of urethral epithelium, and gentle enzymatic digestion to prepare single-cell suspensions. This is the first detailed protocol for the isolation and digestion of female mouse urethral epithelial tissue. The optimized protocol yields an enriched single-cell population of epithelial cells that can be utilized for various downstream analyses. As presented in the study, the authors have performed flow cytometry on a single-cell suspension of urethral epithelial cell isolates, which demonstrates the presence of viable single cells, predominantly of epithelial nature. Further, these epithelial cells are able to generate organoids in 3D culture, which resemble the stratified mouse urethral lining. The authors also demonstrate whole-mount staining of urethral epithelial sheets to study the association of epithelial-associated immune cells, specifically macrophages, with urethral epithelial cells. The methods described in this paper will aid in research on the urethral lining and its role in immune defense.

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an optimized method to isolate female mouse urethral epithelium for single-cell and immunofluorescence analysis

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.

Dissection and collection of the adult female mouse urethra
In the first part of this study, the authors provide a detailed protocol for the dissection of the female mouse urethra. Given its attachment to the ventral vaginal wall and location underneath the pubic symphysis, the method provided here allows for the clean dissection of the full-length female urethra (Figure 1).
Isolation of urethral epithelial tissue and flow cytometry analysis for single-cell isolates
Trypsin digestion results in easy separation of the epithelial lining of the urethra from the surrounding stroma. Epithelial sheets with higher transparency than the stromal tissue can be observed after separation (Figure 2). The separated epithelial sheets yielded single-cell suspensions upon enzymatic digestion. Single cells isolated from the urethral epithelium show consistently high viability (&gt;80%) by trypan blue exclusion on a hemocytometer (Figure 2L) and flow cytometry using viability dyes to gate live cells (Figure 3, Supplementary File 1).
The authors performed flow cytometry to assess epithelial cell enrichment from single-cell suspensions of urethral epithelium. To check cell viability, the cell suspension was stained with a live/dead marker, the amine-reactive ghost dye V450. Authors consistently observed a cell viability of &gt;80% by the live/dead marker. The live/dead marker enabled the gating out of debris and dead cells during flow analysis. They stained samples with an epithelial cell marker (CD326/EPCAM), a macrophage marker (F4/80), and an immune cell marker (CD45) to record the composition of cells isolated through this method. The results of flow cytometry showed that the authors isolated an enriched epithelial cell population from the female mouse urethral epithelium. For both independent experiments performed, the group achieved 80–90 % of EPCAM-positive epithelial cells after gating on live cells. The authors also observed ~1.30 % of CD45+ immune cells in the epithelial preparation. Macrophages associated with the epithelial lining accounted for ~1.18 % of the live cells from the epithelial compartment. Flow cytometry analysis of single-cell suspensions from epithelial sheets shows minimal stromal contamination (5–6%), which is mostly attributed to vascular endothelium according to their previous study5. Supplementary File 1 shows flow cytometry results for single-cell preparations from the epithelial and stromal compartments. Compared to the 80–90% enrichment of epithelial cells isolated from the epithelial compartment, the stromal compartment also showed 40–50% of epithelial cells, which are from the short ducts and glandular epithelium extending from the urethral lumen into the surrounding stroma (Figure 3, Supplementary File 1).
Urethral epithelial organoids culture, histology, and immunostaining
The authors also demonstrate how single epithelial cells isolated from the urethra are viable and can form organoids in a 3D matrix, and observed organoid growth from single urethral epithelial cells up to Day 6 in culture (Figure 4B–G). They performed hematoxylin-eosin staining (H&amp;E) to observe the histology of organoids (Figure 4H–J). The authors observed two distinct morphologies of urethral epithelial organoids, one being large and hollow, while the other type was smaller and solid. In both cases, the organoids were stratified and recapitulated markers expressed in the epithelial lining of the female urethra, including KRT5 and P63 marking the basal layer and KRT4 and KRT13 labeling suprabasal/intermediate cell layers (Figure 4K–N), Supplementary Figure 1). The differences in organoid formation capacity between the proximal and distal urethra were examined. Organoids generated from the proximal urethra had a higher proportion of hollow organoids, while organoids from the distal urethra formed solid spheres (Figure 4O–P). Additionally, they observed differences in organoid-forming efficiency in epithelial cells isolated from the proximal and distal parts of the urethra. Distal urethral epithelium formed organoids with higher efficiency than proximal urethral epithelium with the same initial seeding density (5000 cells/droplet) (Figure 4Q–S).
Whole-mount immunostaining of isolated urethral epithelial sheets
The researchers also present an optimized method for whole-mount staining of the female mouse urethral epithelial sheets. Here, they stained epithelial sheets with two markers: F4/80 for macrophages and E-cadherin/CDH1 to mark epithelial cell-cell junctions (Figure 5). Tissues were imaged using a Zeiss Axio Observer 7 microscope with Apotome 3 optical sectioning. Epithelial sheets show strong labeling with the CDH1 antibody. Whole-mount preparations of urethral epithelium labeled with antibodies to F4/80 showed macrophages forming an arbor of dendrites in the epithelial lining. Macrophages could be observed sending dendritic processes between epithelial cells of the urethra. Overall, the results demonstrate the utility of the protocols described here for the study of the epithelial lining of the female mouse urethra.
<img alt="Mouse dissection sequence; anatomical study; uterus and bladder exposure; scientific analysis." class="xfigimg" src="/files/ftp_upload/70973/70973fig1.jpg" data-altupdatedat="1785816036000" data-alt="Figure 1"> 
Figure 1: Mouse dissection for female urethra collection. (A) Place the mouse in a supine position and spray the abdomen with 70% EtOH. (B–C) Open the abdomen by lifting up the skin and making a vertical incision. (D) Trim fat and connective tissue to expose the butterfly-shaped pubic bones connected at the (Arrow in black) pubic symphysis. (E) Cut the pubic symphysis. (F) Once the pubic bones are removed, the urethra can be seen running downward (Outlined with a dotted line in black) from the bladder. (G) Gently pull up the bladder with forceps. The urethra is clearly visible as a thin red tube. (H–I) Pull the bladder upwards from the body cavity. Use tension on the bladder to separate the urethra from the ventral vaginal wall. Run the scissors between the urethra and the vaginal wall to separate the urinary and reproductive tracts. (J–K) Detach the distal end of the urethra attached to the skin. (L) Image of a dissected female mouse lower urinary tract (FLUT), showing bladder and urethra. <a href="https://www.jove.com/files/ftp_upload/70973/70973fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Enzymatic digestion and separation of urethral tissue into epithelial and stromal compartments, diagram." class="xfigimg" src="/files/ftp_upload/70973/70973fig2.jpg" data-altupdatedat="1785816036000" data-alt="Figure 2"> 
Figure 2: Isolation of urethral epithelium and preparation of a single-cell suspension. (A) Schematic depicting enzymatic digestion and mechanical separation of the epithelial and stromal compartments of the urethra followed by single-cell digestion. (B) Clear the extra fat from the female lower urinary tract. (C–F) Cut the urethra from the bladder neck region to separate urethra from bladder. Then cut urethra in two halves using fine scissors. (G) Wash urethral pieces with 1x HBSS solution by passing over a 100-micron filter. (H) Transfer urethral tissue fragments into 1% trypsin solution. Incubate on a sample rotator in the cold room. Add MgCl₂ and DNase I to the same tube. Incubate at 37 °C in a hybridization oven on a sample rotator. (I–J) Isolate the epithelium by gently squeezing with fine forceps along the length of the urethral fragment. (K) Tissues are minced into small fragments using fine scissors. Transfer the minced epithelial tissue into Accutase digestion solution and incubate at 37 °C in a hybridization oven on a sample rotator. (L) Count epithelial cells and assess viability using trypan blue with a hemocytometer. <a href="https://www.jove.com/files/ftp_upload/70973/70973fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Flow cytometry analysis; scatter plots of urethral epithelium and stroma; CD45, CD326 markers." class="xfigimg" src="/files/ftp_upload/70973/70973fig3.jpg" data-altupdatedat="1785816036000" data-alt="Figure 3"> 
Figure 3: Flow cytometry to assess single-cell suspensions from urethral epithelium and stroma compartments. Flow cytometry was performed on single-cell preparations of pooled mouse urethra (A) epithelial and (B) stromal compartments. Live cells were gated to exclude dead cells stained with Ghost Dye V450. Live cells were assessed for CD45 (pan-immune marker), CD326/EPCAM (pan-epithelial marker) and F4/80 (murine macrophage marker) expression. Data is representative of 2 independent experiments conducted on a pool of 5–6 mice per experiment to generate epithelial and stromal samples. Abbreviations; SSC-A = side scatter-peak area; FITC = fluorescein isothiocyanate; APC = allophycocyanin; PE = phycoerythrin. <a href="https://www.jove.com/files/ftp_upload/70973/70973fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Urethra organoid formation process; mouse cell embedding, trypsin digestion; microscopy images, graphs." class="xfigimg" src="/files/ftp_upload/70973/70973fig4v3.jpg" data-altupdatedat="1785816036000" data-alt="Figure 4"> 
Figure 4: Histology and immunostaining of urethral epithelial organoids. (A) Schematic showing workflow for the generation of urethral epithelial organoids. (B–G) Representative bright-field micrographs of urethral epithelial organoids (UEOs) generated from single cells at different days of in vitro expansion. Representative bright-field images of (H) hollow and (I) solid urethral epithelial organoids (UEOs) stained with hematoxylin and eosin (H&amp;E). (J) Quantification of organoid types generated across three independent experiments. The error bar represents the standard error of the mean. Representative images of (K) hollow and (L) solid urethral epithelial organoids (UEOs) immunostained with epithelial markers KRT13 (suprabasal epithelial marker), KRT5 (basal epithelial marker) and P63 (basal epithelial marker). Images depicting single fluorescent channels are depicted in K’,K’’,K’’’,L’,L’’ and L’’’. Representative images of (M) hollow and (N) solid urethral epithelial organoids (UEOs) immunostained with epithelial markers KRT4 (suprabasal epithelial marker) and KRT5 (basal epithelial marker). Images depicting single fluorescent channels are depicted in M’,M’’,N’ and N”. (O) Schematic of dissection of proximal and distal urethra. (P) Quantification of organoid types generated from the proximal and distal urethra across two independent experiments. Representative bright-field micrographs of Day 6 urethral epithelial organoids (UEOs) generated from epithelium isolated from the (Q) proximal and (R) distal urethra. Data of organoids generated from the complete urethra is representative of 3 independent experiments conducted with a pool of 3–5 mice per experiment. (S) Data of organoids generated from the proximal and distal urethra are representative of 2 independent experiments conducted with a pool of 3–4 mice per experiment. Nuclei labeled with DAPI (blue). (Scale bars, 100 µM). Schematics prepared with BioRender and Adobe Illustrator. Created in BioRender. Binoy joseph, D. (2026) https://BioRender.com/jvf17v7 <a href="https://www.jove.com/files/ftp_upload/70973/70973fig4largev3.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="E-cadherin+F4/80 immunostaining diagram; epithelial separation; microscopy; cell analysis; imaging." class="xfigimg" src="/files/ftp_upload/70973/70973fig5.jpg" data-altupdatedat="1785816036000" data-alt="Figure 5"> 
Figure 5: Whole-mount immunostaining of urethral epithelial sheets. (A) 10-micron frozen section of the adult female mouse urethra in optimal cutting media, showing immunostaining for the murine macrophage marker F4/80 (in red) and the epithelial protein E-cadherin/CDH1 (in green). (A’) represents zoomed in view of inset in (A). (B) Schematic diagram showing steps for whole-mount staining of isolated urethral epithelial sheets. Wholemount immunostaining of isolated female mouse urethral epithelial lining was performed with antibodies against the murine macrophage marker F4/80 (in red) and the epithelial protein E-cadherin/CDH1 (in green). (C) Representative image of merged view. (D) Orthogonal maximal intensity projection of the red channel showing macrophage organization. (E–F) Zoomed in view of epithelium and associated macrophages. White scale bar, 50 µM. Grey scale bar, 500 µM. Data is representative of 2 independent experiments conducted on a pool of 4–5 mice. Schematics prepared in BioRender and Adobe Illustrator. Created in BioRender. Binoy joseph, D. (2026) https://BioRender.com/tjh9opk <a href="https://www.jove.com/files/ftp_upload/70973/70973fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Table 1: Preparation of 1x HBSS (no Ca, Mg).<a href="https://www.jove.com/files/ftp_upload/70973/Supplementary%20Table%201%20(9).xlsx" target="_blank">Please click here to download this file.</a>
Supplementary Table 2: Preparation of 1x sHBSSa and 1 % Trypsin solution.<a href="https://www.jove.com/files/ftp_upload/70973/Supplementary%20Table%202%20(5).xlsx" target="_blank">Please click here to download this file.</a>
Supplementary Table 3: Preparation of cell/flow staining buffer.<a href="https://www.jove.com/files/ftp_upload/70973/Supplementary%20Table%203%20(4).xlsx" target="_blank">Please click here to download this file.</a>
Supplementary Table 4: Preparation of organoid expansion media for urethral epithelial organoids.<a href="https://www.jove.com/files/ftp_upload/70973/Supplementary%20Table%204%20(1).xlsx" target="_blank">Please click here to download this file.</a>
Supplementary Table 5: Preparation of Horse Blocking Buffer.<a href="https://www.jove.com/files/ftp_upload/70973/Supplementary%20Table%205%20(1).xlsx" target="_blank">Please click here to download this file.</a>
Supplementary File 1: Cell counts for flow cytometry experiment performed on single cells from the urethral epithelial and stromal compartments from two independent experiments.<a href="https://www.jove.com/files/ftp_upload/70973/Supplementary%20File%201_Flow%20Experiment%20Data.xlsx" target="_blank">Please click here to download this file.</a>
Supplementary Figure 1: Comparison of adult female mouse urethral epithelium to urethral epithelial organoids. Representative images of (A) adult female mouse urethral epithelium, (B) hollow urethral epithelial organoids, and (C) solid urethral epithelial organoids (UEOs) immunostained with epithelial markers KRT13 (suprabasal epithelial marker), KRT5 (basal epithelial marker), and P63 (basal epithelial marker). Images depicting single fluorescent channels are depicted in A’,A’’,A’’’,B’,B’’,B’’’,C’,C’’ and C’’’. Representative images of (D) adult female mouse urethral epithelium, (E) hollow urethral epithelial organoids and (F) solid urethral epithelial organoids (UEOs) immunostained with epithelial markers KRT4 (suprabasal epithelial marker) and KRT5 (basal epithelial marker). Images depicting single fluorescent channels are depicted in D’,D’’,E’,E’’,F’ and F”. Data representative of 3 independent experiments conducted with a pool of 3–4 mice per experiment. Nuclei labeled with DAPI (blue). (Scale bars, 100 µM).<a href="https://www.jove.com/files/ftp_upload/70973/Supplementary%20Figure%201_Urethra%20and%20UEOs_A4.pdf" target="_blank">Please click here to download this file.</a>

This protocol presents an optimized method for isolating the epithelial lining of the adult female mouse urethra for downstream analyses, including flow cytometry, immunostaining, and organoid culture. Methods yielded enriched, viable urethral epithelial cells, as evidenced by flow cytometry analysis, whole-mount imaging, and efficient organoid formation.

The urethra, lined by epithelial cells, serves as the conduit for urine outflow from the body. The epithelial lining of the urethra comprises distinct ...

Our group is interested in studying epithelial patterning in the genitourinary tract. This protocol represents an optimized method to isolate the epithelial lining of the female mouse urethra for downstream molecular and cellular analysis. The methods described in this paper will aid in research on the female urethral lining and its role in immune defense.To begin, place the euthanized female CD1 mouse 6 to 12 weeks of age in a supine position on the dissection table. Spray 70%ethanol on the abdomen to wet the hair. Using forceps, lift the skin on the abdomen.Then with small scissors, make a vertical incision toward the lower body, ending at the external urethral opening. Identify the bladder and gently lift it with forceps, ensuring it is empty and free of urine. Using scissors, trim the fat and connective tissue around the bladder to expose the butterfly-shaped structure where the two pubic bones join at the pubic symphysis.Observe the urethra running beneath the pubic symphysis. Then lift the pubic symphysis with forceps and cut it open using scissors. Remove the pubic bone fragments to expose the thin urethra lying on the ventral surface of the vaginal wall.Using fine forceps, gently pull the bladder upward from the body cavity. Run scissors between the urethra and the vaginal wall to separate the urinary and reproductive tracts. Maintain slight tension on the bladder while cutting along the back of the urethra to separate it from the vaginal wall.Continue cutting to the distal urethral opening to obtain the full length mouse urethra, measuring 1.2 to 1.5 centimeters in length. Next, separate the urethra from the bladder by making a single cut at the bladder neck. Pool four to five urethras in a sterile Petri dish containing fresh HBSS free of calcium and magnesium.Under a dissecting microscope, trim the fat and connective tissue surrounding the urethra using fine point forceps and scissors. Then with scissors, cut each urethra crosswise into two equal sized pieces. Place the tissue pieces on top of a 100 micron nylon mesh strainer.Wash the tissue pieces with one milliliter of HBSS, then transfer them into a 1.5 milliliter microcentrifuge tube containing one milliliter of 1%trypsin prepared in HBSS supplemented with antibiotics and antimicotics. Next, incubate the sample on a rotator at 30 revolutions per minute and four degrees Celsius for 15 to 30 minutes. After digestion, add 10 millimolar magnesium chloride prepared in HBSS to the tube, followed by 10 micrograms per milliliter of DNAse I prepared in PBS.Incubate the tube for 15 to 30 minutes on a rotator placed inside a 37 degrees Celsius hybridization oven. To quench the trypsin, transfer the urethral tissues into a 1.5 milliliter microcentrifuge tube containing 10%sheep serum in HBSS at room temperature. After five minutes, transfer the urethral tissues into a new tube containing ice cold HBSS, and then into a sterile Petri dish containing ice cold HBSS.While viewing the sample under a microscope, hold one end of the urethral piece with a pair of forceps. Using another pair of forceps, pass along the entire length of the urethral piece to extrude the transparent epithelial sheet. Process some of the isolated urethral epithelial sheets directly for whole mount immunostaining to visualize epithelial cells and epithelial-associated macrophages in situ.Use a small plastic pipette to transfer the epithelial pieces in a drop of HBSS isolated from four to five pooled mouse urethras into a sterile Petri dish. Use scissors to mince the epithelium into small fragments. Transfer the minced epithelial fragments into a two milliliter microcentrifuge tube containing 1.5 milliliters of pre-warmed Accutase digestion solution at 37 degrees Celsius.Incubate the sample on a rotator at 37 degrees for one hour. After incubation, transfer the entire contents into a five milliliter centrifuge tube containing two milliliters of wash buffer composed of calcium and magnesium-free 1X PBS and 1%BSA. Centrifuge the suspension at 600 G for five minutes at four degrees Celsius using a swinging bucket rotor.Next, aspirate the supernatant using a gentle vacuum. Then use a one milliliter filter tip pipette to resuspend the pellet in one milliliter of wash buffer. Break up the pellet by pipetting up and down until the clumps become smaller.Pass the suspension through 23.5 gauge and 26.5 gauge needles, each attached to a one milliliter syringe using four strokes for each needle. Next, add one milliliter of wash buffer to wet the surface of a 30 micron pre-separation filter placed over a five milliliter tube. Using the syringe connected to the 26.5 gauge needle, transfer one milliliter of the cell suspension through the filter.Then wash the tube with one milliliter of wash buffer and pass it through the filter. Pellet the cells by centrifugation at 600 G for five minutes at four degrees Celsius using a swinging bucket rotor. Aspirate the supernatant.Resuspend the pellet in 100 microliters of wash buffer and add 100 microliters of red blood cell lysis buffer. After exactly three minutes, add four milliliters of wash buffer and mix. Harvest the cells by centrifugation at 600 G for five minutes at four degrees Celsius using a swinging bucket rotor.Remove the supernatant prior to resuspending the pellet in 50 to 200 microliters of wash buffer depending on the pellet size. Mix a five microliter aliquot of cells with five microliters of trypan blue and load the mixture onto a hemocytometer to assess cell yield and viability. Assess the cell viability and quantify distinct cell populations using flow cytometry.Culture urethral epithelial single cells in a basement membrane matrix and organoid expansion media to generate three-dimensional urethral epithelial organoids. Perform hematoxylin eosin staining and fluorescent immunostaining on embedded organoid sections to visualize organoid structure and cellular composition. The isolated urethral epithelial sheaths had a higher transparency than the stromal tissue after separation.Whole mount immunostaining of the epithelial sheaths showed strong labeling with the E-cadherin CDH1 antibody. Labeling the sheets with the antibody to F480 showed the macrophages forming branching dendritic structures between the epithelial cells of the urethra. Single cells isolated from the urethral epithelium showed consistently high viability by trypan blue exclusion on a hemocytometer and flow cytometry using the amine reactive Ghost Dye V 450.The isolated cells contained an enriched epithelial cell population after gating on the live cells. Approximately 1.3%of the CD45 positive immune cells and 1.18%macrophages were observed in the single cell suspensions. Single epithelial cells isolated from the urethra remained viable and formed organoids in a three-dimensional matrix with organoid growth observed up to day six in culture.Hematoxylin eosin staining showed two distinct morphologies of the urethral epithelial organoids, including large hollow organoids and smaller solid organoids. Both the hollow and the solid urethral epithelial organoids were stratified and expressed KRT5 and P63 in the basal layer. They expressed KRT-13 in the suprabasal and intermediate cell layers.The organoids generated from the proximal urethra showed a higher proportion of hollow organoids while the organoids from the distal urethra formed more solid spheres. This protocol allows researchers to study urethral cell types and self-renewable capacity of epithelial cells from the proximal and distal urethra. One of the most important considerations of this protocol is the careful dissection of full-length urethra and optimal enzymatic digestion to obtain viable urethral epithelial cells for downstream analysis.The epithelial cells produced by this protocol can be used for multiple applications, including single-cell RNA sequencing, bulk RNA sequencing, flow cytometry, immunostaining, and organoid culture.

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