Interrogating Cell-Cell Interactions in the Salivary Gland <em>via Ex Vivo</em> Live Cell Imaging

Sonia Elder; Justyna Cholewa-Waclaw; Elaine Emmerson

doi:10.3791/65819

JoVE Journal > Immunology and Infection

Inmunología y contagio

Interrogating Cell-Cell Interactions in the Salivary Gland via Ex Vivo Live Cell Imaging

Published: November 17, 2023

doi:

10.3791/65819

Sonia Elder¹, Justyna Cholewa-Waclaw¹, Elaine Emmerson¹

¹The Centre for Regenerative Medicine, Institute for Regeneration and Repair,The University of Edinburgh

Summary

Immunofluorescent imaging is constrained by the ability to observe complex, time-dependent biological processes in just a single snapshot in time. This study outlines a live-imaging approach conducted on precision-cut mouse submandibular gland slices. This approach allows for the real-time observation of cell-cell interactions during homeostasis and the processes of regeneration and repair.

Abstract

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal role played by macrophages in driving the regenerative response. However, our understanding of this critical role has primarily relied on static views obtained from fixed tissue biopsies. To overcome this limitation and gain insights into these interactions in real time, this study outlines a comprehensive protocol for culturing salivary gland tissue ex vivo and capturing live images of cell migration.

The protocol involves several key steps: First, mouse submandibular salivary gland tissue is carefully sliced using a vibratome and then cultured at an air-liquid interface. These slices can be intentionally injured, for instance, through exposure to radiation, to induce cellular damage and trigger the regenerative response. To track specific cells of interest, they can be endogenously labeled, such as by utilizing salivary gland tissue collected from genetically modified mice where a particular protein is marked with green fluorescent protein (GFP). Alternatively, fluorescently-conjugated antibodies can be employed to stain cells expressing specific cell surface markers of interest. Once prepared, the salivary gland slices are subjected to live imaging using a high-content confocal imaging system over a duration of 12 h, with images captured at 15 min intervals. The resulting images are then compiled to create a movie, which can subsequently be analyzed to extract valuable cell behavior parameters. This innovative method provides researchers with a powerful tool to investigate and better understand macrophage interactions within the salivary gland following injury, thereby advancing our knowledge of the regenerative processes at play in this dynamic biological context.

Introduction

Macrophages have been shown to play increasingly important roles in the processes of regeneration and repair, extending beyond their classical immune function¹^,². Indeed, macrophages are involved in a plethora of processes related to regeneration, exhibiting critical regulatory activity in all stages of repair, as well as scar formation and fibrosis³^,⁴. Tissue-resident macrophages are highly heterogeneous cell types with complex mechanisms driving diverse cellular phenotypes, and they play essential roles in organ development, function, and homeostasis (as reviewed in⁵). Tissue-resident macrophages initially arise from precursors in the yolk sac and fetal liver, and they are subsequently replaced by proliferation or by bone marrow-derived blood monocytes at varying rates, depending on the longevity of existing macrophages and the tissue or niche within which they reside⁶^,⁷.

Importantly, tissue-resident macrophages are dispersed throughout all tissues and contribute to diverse organ functions. They are uniquely programmed by their microenvironment to perform niche-specific functions. For this reason, the localization of macrophages within the tissue offers insight into their function, with unique populations observed in the lung, mammary gland, intestine, skin, and muscle⁸^,⁹^,¹⁰. During mammary gland development, ductal macrophages are intimately associated with the ductal tree, and their depletion results in significantly reduced branching¹¹. Furthermore, macrophages are required for morphogenesis during puberty and alveologenesis in pregnancy, where they actively monitor the epithelium. In muscle injury, a specific population of macrophages "dwells" within the injury site, providing a transient niche in which they supply proliferation-induced cues required for stem cell proliferation. Thus, they exhibit the specialized role of distinct macrophage populations in governing the repair process². In the lung, a similar phenomenon occurs where interstitial macrophages prime interleukin (IL)-R1-expressing alveolar type II (AT2) cells for conversion into damage-associated transient progenitors through the release of IL-1B¹². Furthermore, recent research has shown that macrophages are essential for the regeneration of the mouse submandibular salivary gland (SMG) after irradiation injury, and in their absence, epithelial regeneration is disrupted¹³. Taken together, this data highlights the importance of macrophage activation and function in transient inflammatory niches after tissue injury, as well as during homeostasis.

Macrophages are active cells, and their functions involve interactions with a variety of different cell types, including direct cell-cell contact¹⁴^,¹⁵, as well as more indirect methods such as the secretion of soluble factors²^,¹⁶, which are essential for niche regulation. While classical immunofluorescent imaging is useful to begin unraveling these interactions, it is limited by depicting only a single snapshot in time, thereby omitting numerous timepoints critical to a highly dynamic regenerative process¹⁷^,¹⁸. As the importance of timing and the emergence of different waves of regeneration come into sharper focus, it is essential to dissect these processes in greater detail.

Radiotherapy is a life-saving treatment for many people diagnosed with cancer. While often effective at shrinking or eliminating the tumor(s), radiotherapy can also damage healthy tissues lying in the radiation field and elicit an immune response. Radiation injury can induce rapid macrophage recruitment, and direct and indirect immunomodulatory responses¹⁹^,²⁰. The salivary glands are often inadvertently irradiated during treatment for head and neck cancer²¹^,²², leading to epithelial damage, cell atrophy, and fibrosis²³^,²⁴, resulting in xerostomia or chronic dry mouth²⁵.

The salivary gland is composed of a plethora of cell types and structures, including but not limited to epithelial cells (both saliva-producing acinar cells and saliva-transporting ductal cells), myoepithelial cells, epithelial progenitor cells, nerves, blood vessels, immune cells, fibroblasts, and extracellular matrix (ECM). The role and response of many of these cell types in the regenerative response have been previously described²⁶^,²⁷^,²⁸^,²⁹^,³⁰. However, how these different cells interact during homeostasis and regeneration, and particularly how immune cells such as macrophages behave, is less well studied. This manuscript describes a newly established method to study the live interactions between SMG macrophages and other cells of interest in ex vivo tissue. The SMG is sliced on a vibratome, stained for surface markers, and imaged for up to 12 h. Using this method, phagocytosis of surrounding cells by macrophages can be observed, macrophage migration kinetics can be studied, and direct cell-cell interactions between macrophages and epithelial cells can be demonstrated.

Protocol

All procedures were approved by the UK Home Office and performed under PPLs PB5FC9BD2 and PP0330540. All experiments align to ARRIVE guidelines and those of the University of Edinburgh.

Wild-type mice were commercially obtained (see Table of Materials). Krt14^CreER;R26^mTmGmice were bred in house, by crossing Krt14^CreER/+mice³¹ with R26^{mTmG/ mTmG} mice³². In all experiments, female 8-10 week old mice were used.

1. Collecting and embedding the SMG

Euthanize the mouse/mice by exposing them to a rising concentration of carbon dioxide (CO₂). Afterward, spray the incision area with 70% ethanol (EtOH) and use scissors and forceps to carefully dissect the SMG(s) from the surrounding tissue (Figure 1A). Remove any fat and connective tissue using forceps, and place the gland in a collection tube containing ice-cold Hanks Balanced Salt Solution (HBSS, see Table of Materials).
Separate the gland into pieces measuring approximately 1 cm² each using forceps.
Heat 4% agarose to 50 °C, and then pour it directly into a 35 mm dish.
Pour a small amount of melted 50 °C agarose into a separate dish and thoroughly rinse the gland by moving it around in the excess agarose.
NOTE: This step is necessary to remove excess liquid from the glands. An excess of liquids can prevent the glands from adhering properly to the agarose.
Position 4-6 pieces of the gland into the agarose, ensuring that they lie flat and are in the same plane (Figure 1A).
NOTE: Ensure the glands do not touch the surface of the agar or the bottom of the plate.
Place the lid on the 35 mm dish and transfer it to an icebox, covering the plate with ice.
Wait for 5-10 min for the agarose to solidify.

2. Sectioning

Use a scalpel to carefully cut around the gland-embedded agarose block, removing the block containing the tissue from the dish. Leave approximately a 5 mm border.
Attach the agarose block to the stage of the vibratome (see Table of Materials) by applying a drop of superglue, ensuring that the entire bottom surface of the block is in contact with the superglue.
Allow the superglue to harden for approximately 5 min. Then, fill the vibratome chamber with ice-cold 1x phosphate-buffered saline (PBS) containing 1% Penicillin-Streptomycin (P/S).
Add ice to both the side and bottom of the vibratome chamber.
NOTE: Maintain a very cold environment and replace the ice as needed.
Trim off any excess agarose and create 5 mm gaps between each piece of gland by making incisions with a scalpel. This will yield individual slices.
Align the vibratome blade with the face of the agarose block and set the start and end points of the sections to achieve the desired slice thickness.
Before starting the cutting process, ensure that the blade is not in contact with the block. This precaution will prevent accidentally cutting large pieces of agarose and losing parts of the sample.
Cut sections with a thickness of 150 µm at a low speed and high vibration (e.g., a speed of 3 and a vibration setting of 8-10 on a scale of 1-10 for each parameter) (Figure 1A).
Once a section is cut and released into the surrounding PBS, collect the sections using a paintbrush and place them in a dish containing pre-warmed Roswell Park Memorial Institute (RPMI) media with P/S.

3. Culturing and staining of SMG slices

Culturing
1. Add 1.5 mL of RPMI media to each well of a 6-well plate that contains a 0.4 µm filter. Ensure there are no air bubbles trapped underneath the filter.
2. Transfer 1-6 slices onto each filter using a paintbrush, taking care not to break the slices.
  NOTE: Ensure that the slices are not submerged in media but are instead floating on the filter. If they are fully submerged, they may suffocate during culture.
3. Incubate at 37 °C with 5% CO₂, and change the media every 3 days (Figure 1A).
4. Irradiate experimental plates with a single dose of 10 Gy gamma radiation using a commercially available irradiator (see Table of Materials). Afterward, return them to the incubator.
Staining for live imaging
1. Using a paintbrush, gently lift the slices from the filter and place them into a 24-well plate containing 500 µL of culture media per well, where the slices will be submerged.
2. Add the relevant conjugated antibodies and nuclear stain (see Table of Materials) to the media at the appropriate concentrations²⁷.
3. Incubate the slices with antibodies for 2 h at 37 °C with gentle agitation.
4. Wash the slices by submerging them in culture media and washing for 3 rounds of 10 min washes at 37 °C with gentle agitation.

4. Mounting and live imaging

Using forceps, remove the tape from one side of a double-sided imaging spacer and adhere it, adhesive side down, to the bottom of a glass-bottom 6-well plate.
Pipette 50 µL of media into the gap in the center of the spacer.
Place the slice into the media using a paintbrush, ensuring that it lies flat without any trapped air bubbles.
Using a pipette, carefully remove 20 µL of media from the gap.
Remove the tape from the top side of the spacer using forceps and place a 25 mm circular coverslip on top. Press around the edge of the spacer to ensure that the coverslip firmly attaches, being cautious not to break the coverslip by applying too much force (Figure 1A).
Image the slice on a confocal microscope for the desired time period or intervals (e.g., every 15 min for 12 h using a 20x water objective).

Representative Results

The response of macrophages to injury in the submandibular salivary gland remains unknown. This includes whether they localize and migrate to specific structures within the gland, as well as the distance and speed at which they migrate. This has been difficult to determine through static imaging approaches.

To address this, a live imaging approach to study macrophage-epithelial cell interaction in real-time has been developed. Immunofluorescent staining of slices was combined with an endogenously labeled lineage-tracing mouse model (Figure 1A and Figure 2A). In this model, slices were exposed to 10 Gy of gamma irradiation to induce irradiation injury and were imaged at 2 h, 3 days, and 4 days post-irradiation (IR), with non-irradiated slices serving as controls. Data were acquired from four channels: Hoechst (using laser 425 to minimize phototoxicity), GFP (488), dTomato (561), and Alexa 647 (640). A z-stack was performed, and an image was captured every 2-3 µm, with a total of 30 to 40 planes collected, resulting in an overall height of 80-90 µm.

Using this technique and analyzing membrane-bound Tomato (mT) signal and Caspase3/7 activity, it became evident that organotypic slices retained their epithelial structure, survived in culture, and exhibited minimal cell death. The data showed that non-irradiated slices of the submandibular gland from R26^mTmG mice³², cultured for 7 days, retained their mT signal and epithelial architecture (Figure 1B). However, at 3 days following ex vivo irradiation, acinar and ductal cell atrophy was evident (Figure 1B; acini and ductal structures highlighted by dashed white and green lines, respectively), consistent with in vivo radiation injury¹³. Apoptosis was negligible in the non-irradiated slices, but there was evidence of Caspase 3/7⁺ cells in the irradiated slices at 4 days post-irradiation (Figure 1C; green arrows), similar to in vivo injury³³^,³⁴. Furthermore, irradiated slices recapitulated in vivo DNA damage³⁴^,³⁵^,³⁶^,³⁷, as indicated by elevated γH2AX³⁸ compared to non-irradiated controls (Figure 1D,E). Thus, organotypic salivary gland slices exhibited good viability in culture and responded similarly to in vivo tissue to irradiation.

Following this, real-time cell-cell interactions were investigated. Macrophages directly interacting with GFP-labeled epithelial progenitor cells (Krt14^CreER-GFP) were observed over a 12 h time period at 3 days post-IR (Figure 2A and Video 1). Remarkably, it was evident that macrophages remained in close proximity to epithelial progenitor cells for hours, often staying in contact for the entire 12 h imaging period. Furthermore, real-time phagocytosis of epithelial cells by macrophages was observed, confirming that macrophages carry out their traditional function in the slice culture model (Figure 2B, Videos 2 and Video 3). This technique also identified that macrophages are relatively stationary during both homeostasis and following irradiation injury, likely due to their high density within the tissue. This demonstrates, for the first time, that salivary gland macrophages do not migrate extensively during homeostasis or after irradiation injury. However, while macrophages did not show significant migration, the surrounding tissue displayed increased dynamics following irradiation, with multiple macrophages actively interacting around clusters of labeled epithelial cells. Additionally, based solely on nuclear staining, this technique allowed us to visualize cell movement within the slice over time, often with entire ducts appearing to 'migrate' within the tissue (Video 4). Over time during the culturing process, it became evident that slices shifted from a flat to a spheroid-like morphology, suggesting that cell movement within the slice is likely due to their rearrangement into a sphere-like structure, resembling a potential reorganization event.

Finally, the live imaging data generated by this assay can be used to quantitatively measure cell behavior, such as migration. Individual cells can be detected and segmented (Figure 3A), and migration can be measured using a commercially available imaging and analysis software (see Table of Materials and Video 5). Furthermore, nearest object analysis can be undertaken to determine whether cells, in this case, macrophages, migrate closer to other cells of interest, in this case, Krt14^CreER-GFP+ cells, and how this dynamic changes over time (Figure 3B).

Figure 1: Recapitulation of in vivo response in precision-cut salivary gland tissue slices. (A) Schematic of the experimental protocol. (B) Representative images of membrane-bound Tomato obtained from fresh submandibular gland (SMG) tissue, unmanipulated SMG slices cultured for 7 days, or SMG slices irradiated with a single dose of 10 Gy gamma irradiation 3 or 7 days earlier. Dashed white lines indicate example acinar structures, and dashed green lines indicate example ductal structures. Scale bar = 50 µm. (C) Representative images of Caspase-3/7 expression obtained from unmanipulated SMG slices or SMG slices irradiated with a single dose of 10 Gy gamma irradiation 2 h or 4 days earlier. Green arrowheads indicate positive nuclei. Scale bar = 50 µm. (D) Representative images of γH2AX expression obtained from unmanipulated SMG slices or SMG slices irradiated with a single dose of 10 Gy gamma irradiation 3 days earlier. Scale bar = 20 µm. (E) Representative expression of γH2AX in EpCAM⁺ epithelial cells from unmanipulated SMG slices or SMG slices irradiated with a single dose of 10 Gy gamma irradiation 2 h and 3 days earlier. SSA = side scatter area. Please click here to view a larger version of this figure.

Figure 2: Live imaging of macrophage-epithelial cell interactions and phagocytosis. (A) Sequential still images of interactions between KRT14+ progenitor cells and their progeny (Krt14^CreER-GFP+cells) and macrophages following irradiation. Live cell imaging captures cellular dynamics over a 90 min period. Scale bar = 20 µm. Associated video is Video 1. (B) Sequential still images of a macrophage phagocytosing an epithelial cell. Live cell imaging shows the process over a 60 min period. White arrows point to the macrophage, and yellow arrows indicate the nucleus of the cell undergoing phagocytosis. Scale bar = 50 µm. Associated video is Video 2. Please click here to view a larger version of this figure.

Figure 3: Live imaging for cellular dynamics analysis. (A) Image illustrating individual cell identification and segmentation for analyzing cellular behavior parameters, such as migration (see Video 5). Cells are pseudocolored randomly for individual cell and track distinction. Scale bar = 100 µm. (B) Quantification of the distance (in µm) of the nearest object to individual Krt14^CreER-GFP+cells plotted over 10 time points (images captured every 5 min). Data presented as the mean value per well. Please click here to view a larger version of this figure.

Video 1: Live imaging revealing dynamic interactions between KRT14+ progenitor cells/progeny and macrophages after irradiation. Slices were exposed to a single 10 Gy gamma irradiation dose before live imaging. Krt14^CreER-GFP+cells are represented in green, macrophages (F4/80+) in red, and nuclei in cyan. The video comprises a single z-stack, spanning a 12 h culture period with images captured every 15 min. Please click here to download the video.

Video 2: Live imaging capturing a macrophage engulfing an epithelial cell. Macrophages (F4/80+) are depicted in red, and nuclei are shown in cyan. The video consists of a single z-stack and spans a 12 h culture period with images taken every 15 min.Please click here to download the video.

Video 3: Maximum projection live imaging showing a macrophage engulfing an epithelial cell. Macrophages (F4/80+) are displayed in red, and nuclei are shown in cyan. The video features a maximum projection of z-stack images totaling 80 µm (a single plane is presented in Video 2) and spans a 12 h culture period with images taken every 15 min.Please click here to download the video.

Video 4: Live imaging illustrating the dynamic behavior of salivary gland epithelium in culture after irradiation. Slices were subjected to a single 10 Gy gamma irradiation dose before live imaging. Macrophages (F4/80+) are represented in red, nuclei in cyan. The video comprises a single z-stack, spanning a 12 h culture period with images captured every 15 min.Please click here to download the video.

Video 5: Quantitative cell migration tracking over time. This video demonstrates the individual tracing of cell tracks from live imaging videos. Arrows indicate cell tracks, and cells are pseudo-colored individually. The video consists of a single z-stack and spans a 1 h culture period with images taken every 15 min.Please click here to download the video.

Discussion

The ability to culture salivary gland tissue ex vivo presents an excellent opportunity to study cell-cell interactions in the context of both homeostasis and injury response. Although intravital imaging of the mouse submandibular gland is feasible³⁹^,⁴⁰, this technique depends on using fluorescent reporter mouse models to endogenously label cells of interest and must be performed under terminal anesthesia. Here, a method to culture submandibular gland slices ex vivo is described, maintaining cellular architecture and cell-cell interactions. This approach refines current live imaging techniques and provides an alternative to intravital imaging.

The long-term maintenance of tissue using this technique relies on culturing slices at an air-liquid interface. Previous explant models²⁶^,⁴¹ have likely achieved successful culture for only a few days because they were submerged in media and essentially "suffocated." In contrast, the use of an air-liquid interface culture system maintains tissue health and structure for an extended period, ensuring high-quality imaging. The method of mounting SMG slices before imaging, with a small amount of media and within a space-restricted chamber to keep the slice flat, is integral to the technique's success. Visualization of cells in this assay depends on endogenously labeled reporter mice or fluorescently-conjugated antibodies. The abundance of transgenic fluorescent reporter mouse models and conjugated antibodies targeting specific cell types and subsets makes this method suitable for exploring various cell-specific interactions.

While this method provides a good model of tissue in situ and ex vivo irradiation injury results in acinar and ductal structure atrophy, similar to what occurs in vivo¹³, some elements cannot be recapitulated ex vivo. These include the lack of functioning vasculature and neuronal input, as well as the absence of infiltrating inflammatory cells. Given the well-documented role of blood vessels and nerves in salivary gland homeostasis and regeneration²⁶^,⁴² and the importance of migratory immune cells⁴³, such as T and B cells, in salivary gland function, injury response, infection, and Sjögren's Syndrome (SS) pathogenesis (as reviewed in⁴⁴), this assay may miss some important cellular interactions. Additionally, very fast migratory events, such as Natural Killer (NK) cell⁴⁵ and Dendritic Cell (DC)⁴⁶ movement, may be missed by imaging every 15 min. However, imaging intervals can be optimized to study the specific cell-cell interactions of interest, and the ability to image in 3 dimensions through z-stacks allows the assessment of 3D cell movement. Securely mounting the tissue during imaging is crucial for quantification, such as cell tracking measurements. Furthermore, although this study utilized mouse tissue, the protocol provides a viable method for studying cell-cell interactions in human salivary glands, generating valuable translational information unattainable through other methods.

While the role of tissue-resident macrophages in homeostasis and regeneration has been demonstrated in several tissues²^,¹⁰^,¹¹^,¹², their role in the salivary glands remains largely unanswered. Although it is known that macrophages are essential for epithelial regeneration after irradiation injury¹³, the precise mechanisms underlying this effect remain unknown. Live imaging of salivary gland slices enables real-time visualization and analysis of complex tissue dynamics, which are often missed by traditional confocal imaging. Additionally, it is evident that macrophages undergo dynamic changes in shape while performing various functions in vivo⁴⁷^,⁴⁸^,⁴⁹, and this protocol likely provides a better representation of these changes than a typical static view in fixed tissue. Future studies can utilize this technique to investigate how cell-cell communication changes across the course of homeostasis, injury, and regeneration/resolution. This approach will be useful for elucidating key signaling pathways and events that may ultimately offer therapeutic benefits.

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

SE is funded by Wellcome Trust grant 108906/Z/15/Z; EE is funded by UKRI/MRC grant MR/S005544/1 and by a Chancellor's Fellowship from the University of Edinburgh. Figure 1A is created with BioRender.com.

Materials

0.4 µm filter cell culture inserts (Nunc)	Avantor/VWR	734-2240	Inserts pre-packed in 6-well multidishes, 20 mm × 25 mm
24 well plate	Corning	3524
35 mm dish	Falcon	353001
6 well plate	Corning	3516
Coverslips	Paul Marienfeld GmbH & Co. KG	111650	Deckglaser Cover Glasses 25 mm diameter
Double-sided sticker	Grace Bio-Labs	654004	SecureSeal Imaging Spacers SS1 x 13, 13 mm diameter x 0.12 mm depth, 25 mm x 25mm OD
EtOH	Scientific Laboratories Supplies	CHE1924	Absolute ethanol (EtOH) AR, 99.7%
F4/80 antibody	Invitrogen	17-4801-82	F4/80 Monoclonal Antibody (BM8), APC, eBioscience
Forceps	Fine Science Tools	91113-10	Student Fine Forceps Straight Broad Shanks
Glass bottom 6 well plate	Cellvis	P06-1.5H-N	6 well glass bottom plate with high performance #1.5 cover glass
Hanks Balanced Salt Solution (HBSS)	Life Technologies	14025050	+calcium +magnesium, no phenol red
Hoechst	Sigma Aldrich	14533	Alternative name: bisBenzimide H 33342 trihydrochloride
Ice box	Fisher Scientific	11339623	Azlon Polyurethane Ice Buckets with Lid
Imaging and analysis software	Harmony
Low Melting Agarose	Merck	A9414-25G
Paintbrush			Watercolour brush, 10 mm x 2mm tip
Penicillin-Streptomycin	Sigma Aldrich	P4333	10,000 units penicillin and 10 mg streptomycin/mL, 0.1 μm filtered
Phosphate Buffered Saline (PBS)	Life Technologies	20012027
RPMI	ThermoFisher	12634010	Gibco Advanced DMEM/F-12
Scalpel	Swann-Morton		Disposable scalpels, No. 11 blade
Scissors	Fine Science Tools	14088-10	Extra Narrow Scissors 10.5 cm
Shepherd Mark-I-68A 137Cs irradiator	JL Shepherd & Associates
Superglue	Bostik		Multi-purpose superglue, fast setting, ultra strong
Vibratome	Leica		Leica VT 1000 S
Vibratome blades	Astra		Superior Platinum Double Edge blade
Wild-type (C57BL/BJ) mice	Charles River

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Elder, S., Cholewa-Waclaw, J., Emmerson, E. Interrogating Cell-Cell Interactions in the Salivary Gland via Ex Vivo Live Cell Imaging. J. Vis. Exp. (201), e65819, doi:10.3791/65819 (2023).