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&#39;s Fellowship from the University of Edinburgh. Figure 1A is created with BioRender.com.

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). Krt14CreER; R26mTmG mice were bred in house, by crossing Krt14CreER/+ mice31 with&#160;R26mTmG/ mTmG mice32. In all experiments, female 8-10 week old mice were used.
1. Collecting and embedding the SMG
<ol>
	<li>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).</li>
	<li>Separate the gland into pieces measuring approximately 1 cm2 each using forceps.</li>
	<li>Heat 4% agarose to 50 &#176;C, and then pour it directly into a 35 mm dish.</li>
	<li>Pour a small amount of melted 50 &#176;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.</li>
	<li>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.</li>
	<li>Place the lid on the 35 mm dish and transfer it to an icebox, covering the plate with ice.</li>
	<li>Wait for 5-10 min for the agarose to solidify.</li>
</ol>
2. Sectioning
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>Add ice to both the side and bottom of the vibratome chamber. 
	NOTE: Maintain a very cold environment and replace the ice as needed.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Cut sections with a thickness of 150 &#181;m 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).</li>
	<li>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.</li>
</ol>
3. Culturing and staining of SMG slices
<ol>
	<li>Culturing
	<ol>
		<li>Add 1.5 mL of RPMI media to each well of a 6-well plate that contains a 0.4 &#181;m filter. Ensure there are no air bubbles trapped underneath the filter.</li>
		<li>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.</li>
		<li>Incubate at 37 &#176;C with 5% CO2, and change the media every 3 days (Figure 1A).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Staining for live imaging
	<ol>
		<li>Using a paintbrush, gently lift the slices from the filter and place them into a 24-well plate containing 500 &#181;L of culture media per well, where the slices will be submerged.</li>
		<li>Add the relevant conjugated antibodies and nuclear stain (see Table of Materials) to the media at the appropriate concentrations27.</li>
		<li>Incubate the slices with antibodies for 2 h at 37 &#176;C with gentle agitation.</li>
		<li>Wash the slices by submerging them in culture media and washing for 3 rounds of 10 min washes at 37 &#176;C with gentle agitation.</li>
	</ol>
	</li>
</ol>
4. Mounting and live imaging
<ol>
	<li>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.</li>
	<li>Pipette 50 &#181;L of media into the gap in the center of the spacer.</li>
	<li>Place the slice into the media using a paintbrush, ensuring that it lies flat without any trapped air bubbles.</li>
	<li>Using a pipette, carefully remove 20 &#181;L of media from the gap.</li>
	<li>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).</li>
	<li>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).</li>
</ol>

Real-Time Live Imaging of Mouse Submandibular Gland Regeneration

The authors have no conflicts of interest to disclose.

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&#160;have likely achieved successful culture for only a few days because they were submerged in media and essentially &#34;suffocated.&#34; 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&#39;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&#160;and the importance of migratory immune cells43, such as T and B cells, in salivary gland function, injury response, infection, and Sj&#246;gren&#39;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.

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 &#34;dwells&#34; 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.

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

interrogating cell-cell interactions in the salivary gland via ex vivo live cell imaging

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.

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 &#181;m, with a total of 30 to 40 planes collected, resulting in an overall height of 80-90 &#181;m.
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 R26mTmG 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&#160;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 &#947;H2AX38 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&#160;investigated. Macrophages directly interacting with GFP-labeled epithelial progenitor cells (Krt14CreER-GFP) were&#160;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 &#39;migrate&#39; 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, Krt14CreER-GFP+&#160;cells, and how this dynamic changes over time (Figure 3B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65819/65819fig1.jpg" /> 
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 &#181;m. (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 &#181;m. (D) Representative images of &#947;H2AX 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 &#181;m. (E) Representative expression of &#947;H2AX 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. <a href="https://www.jove.com/files/ftp_upload/65819/65819fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65819/65819fig2.jpg" /> 
Figure 2: Live imaging of macrophage-epithelial cell interactions and phagocytosis. (A) Sequential still images of interactions between KRT14+ progenitor cells and their progeny (Krt14CreER-GFP+&#160;cells) and macrophages following irradiation. Live cell imaging captures cellular dynamics over a 90 min period. Scale bar = 20 &#181;m. 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 &#181;m. Associated video is Video 2. <a href="https://www.jove.com/files/ftp_upload/65819/65819fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65819/65819fig3.jpg" /> 
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 &#181;m. (B) Quantification of the distance (in &#181;m) of the nearest object to individual Krt14CreER-GFP+&#160;cells plotted over 10 time points (images captured every 5 min). Data presented as the mean value per well. <a href="https://www.jove.com/files/ftp_upload/65819/65819fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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. Krt14CreER-GFP+&#160;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.&#160;<a href="https://www.jove.com/files/ftp_upload/65819/Elder et al_Video 1.mp4" target="_blank">Please click here to download the video.</a>
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.<a href="https://www.jove.com/files/ftp_upload/65819/Elder et al_Video 2.wmv" target="_blank">Please click here to download the video.</a>
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 &#181;m (a single plane is presented in Video 2) and spans a 12 h culture period with images taken every 15 min.<a href="https://www.jove.com/files/ftp_upload/65819/Elder at al_Video 3.wmv" target="_blank">Please click here to download the video.</a>
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.<a href="https://www.jove.com/files/ftp_upload/65819/Elder et al_Video 4.wmv" target="_blank">Please click here to download the video.</a>
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.<a href="https://www.jove.com/files/ftp_upload/65819/Elder at al_Video 5.wmv" target="_blank">Please click here to download&#160;the video.</a>

Watch this Scientific Journal Video about Studying Macrophage-Epithelial Cell Interactions in Salivary Gland Regeneration After Injury at JoVE.com

Author Spotlight: Studying Macrophage-Epithelial Cell Interactions in Salivary Gland Regeneration After Injury 

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.

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

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal ...

interrogating-cell-cell-interactions-salivary-gland-via-ex-vivo-live

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JoVE Journal

Immunology and Infection

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This video describes the manipulation of cultured neurons using laser tweezers in vitro.

In this paper and video, we describe the protocols used in our laboratory to study the targeting preferences of regenerating cell processes of adult ...

Biology

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended intracellular endotubes that build and stabilize the lumen with a membrane and submembraneous cytoskeleton of apical character. The excretory cell expands its length approximately 2,000 times to generate these canals, making this model unique for the in vivo assessment of de novo polarized membrane biogenesis, intracellular lumen morphogenesis and unicellular tubulogenesis. The protocol presented here shows how to combine standard labeling, gain- and loss-of-function genetic or RNA interference (RNAi)-, and microscopic approaches to use this model to visually dissect and functionally analyze these processes on a molecular level. As an example of a labeling approach, the protocol outlines the generation of transgenic animals with fluorescent fusion proteins for live analysis of tubulogenesis. As an example of a genetic approach, it highlights key points of a visual RNAi-based interaction screen designed to modify a gain-of-function cystic canal phenotype. The specific methods described are how to: label and visualize the canals by expressing fluorescent proteins; construct a targeted RNAi library and strategize RNAi screening for the molecular analysis of canal morphogenesis; visually assess modifications of canal phenotypes; score them by dissecting fluorescence microscopy; characterize subcellular canal components at higher resolution by confocal microscopy; and quantify visual parameters. The approach is useful for the investigator who is interested in taking advantage of the C. elegans excretory canal for identifying and characterizing genes involved in the phylogenetically conserved processes of intracellular lumen and unicellular tube morphogenesis.

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The C. elegans excretory canal is a unique single-cell model for the visual in vivo analysis of de novo polarized membrane biogenesis. This protocol describes a combination of standard genetic/RNAi and imaging approaches, adaptable for the identification and characterization of molecules directing unicellular tubulogenesis, and apical membrane and lumen biogenesis.

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended ...

Developmental Biology

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

Neuroscience

Live Tracking of Dental Cells via Two-Photon Microscopy

Using Ex Vivo Live Imaging to Investigate Cell Divisions and Movements During Mouse Dental Renewal

The continuously growing mouse incisor is emerging as a highly tractable model system to investigate the regulation of adult epithelial and mesenchymal stem cells and tooth regeneration. These progenitor populations actively divide, move, and differentiate to maintain tissue homeostasis and regenerate lost cells in a responsive manner. However, traditional analyses using fixed tissue sections could not capture the dynamic processes of cellular movements and interactions, limiting our ability to study their regulations. This paper describes a protocol to maintain whole mouse incisors in an explant culture system and live-track dental epithelial cells using multiphoton timelapse microscopy. This technique adds to our existing toolbox for dental research and allows investigators to acquire spatiotemporal information on cell behaviors and organizations in a living tissue. We anticipate that this methodology will help researchers further explore mechanisms that control the dynamic cellular processes taking place during both dental renewal and regeneration.

Author Spotlight: Understanding Dynamic Cellular Behaviors in Adult Mouse Dental Tissue Renewal and Repairment 

Ex vivo live imaging is a powerful technique for studying the dynamic processes of cellular movements and interactions in living tissues. Here, we present a protocol that implements two-photon microscopy to live track dental epithelial cells in cultured whole adult mouse incisors.

The continuously growing mouse incisor is emerging as a highly tractable model system to investigate the regulation of adult epithelial and mesenchymal ...

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, including cell biology, oncology, dentistry, and immunology. The SMG is an exocrine gland comprised of secretory epithelial cells, myofibroblasts, endothelial cells, nerves, and extracellular matrix. Dynamic cellular processes in the rat and mouse SMG have previously been imaged, mostly using inverted multi-photon microscope systems. Here, we describe a straightforward protocol for the surgical preparation and stabilization of the murine SMG in anesthetized mice for in vivo imaging with upright multi-photon microscope systems. We present representative intravital image sets of endogenous and adoptively transferred fluorescent cells, including the labeling of blood vessels or salivary ducts and second harmonic generation to visualize fibrillar collagen. In sum, our protocol allows for surgical preparation of mouse salivary glands in upright microscopy systems, which are commonly used for intravital imaging in the field of immunology.

We describe a protocol to surgically expose and stabilize the murine submandibular salivary gland for intravital imaging using upright intravital microscopy. This protocol is easily adaptable to other exocrine glands of the head and neck region of mice and other small rodents.

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, ...

Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to restore function of glands damaged by radiation therapies or due to pathologies associated with Sj&#246;gren's syndrome. A second goal of researchers is to define the mechanisms by which viruses replicate within glandular tissue where they can then gain access to salivary fluids important for horizontal transmission. These goals highlight the need for a robust and accessible in vitro salivary gland model that can be utilized by researchers interested in the above mentioned as well as related research areas. Here we discuss a simple protocol to isolate epithelial cells from human salivary glands and propagate them in vitro. Our protocol can be further optimized to meet the needs of individual studies. Briefly, salivary tissue is mechanically and enzymatically separated to isolate single cells or small clusters of cells. Selection for epithelial cells occurs by plating onto a basement membrane matrix in the presence of media optimized to promote epithelial cell growth. These resulting cultures can be maintained as three-dimensional clusters, termed "salispheres", or grown as a monolayer on treated plastic tissue culture dishes. This protocol results in the outgrowth of a heterogenous population of mainly epithelial cells that can be propagated for 5-8 passages (15-20 population doublings) before undergoing cellular senescence.

We present a method for isolating and cultivating primary human salivary gland-derived epithelial cells. These cells exhibit gene expression patterns consistent with them being of salivary epithelial origin and can be grown as salispheres on basement membrane matrices derived from Engelbreth-Holm-Swarm tumor cells or as monolayers on treated culture dishes.

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood 4. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo5. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-1116,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands8. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.

A technique to genetically manipulate epithelial cells within whole ex vivo cultured embryonic mouse submandibular glands (SMGs) using viral gene transfer is described. This method takes advantage of the innate ability of SMG epithelium and mesenchyme to spontaneously recombine after separation and infection of epithelial rudiments with adenoviral vectors.

Branching  morphogenesis occurs during the development of many organs, and the embryonic  mouse submandibular gland (SMG) is a classical model for the ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Intravital Subcellular Microscopy of the Mammary Gland

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory mechanisms. During pregnancy, the gland expands from a primitive ductal tree embedded in a fat pad to a highly branched alveolar network primed for the formation and secretion of colostrum and milk. Post-partum, the gland supplies all the nutrients required for neonatal survival, including membrane-coated lipid droplets (LDs), proteins, carbohydrates, ions, and water. Various milk components, including lactose, casein micelles, and skim-milk proteins, are synthesized within the alveolar cells and secreted from vesicles by exocytosis at the apical surface. LDs are transported from sites of synthesis in the rough endoplasmic reticulum to the cell apex, coated with cellular membranes, and secreted by a unique apocrine mechanism. Other preformed constituents, including antibodies and hormones, are transported from the serosal side of the epithelium into milk by transcytosis. These processes are amenable to intravital microscopy because the mammary gland is a skin gland and, therefore, directly accessible to experimental manipulation. In this paper, a facile procedure is described to investigate the kinetics of LD secretion in situ, in real-time, in live anesthetized mice. Boron-dipyrromethene (BODIPY)665/676&#160;or monodansylpentane are used to label the neutral lipid fraction of transgenic mice, which either express soluble EGFP (enhanced green fluorescent protein) in the cytoplasm, or a membrane-targeted peptide fused to either EGFP or tdTomato. The membrane-tagged fusion proteins serve as markers of cell surfaces, and the lipid dyes resolve LDs &#8805; 0.7 &#181;m. Time-lapse images can be recorded by standard laser scanning confocal microscopy down to a depth of 15-25 &#181;m or by multiphoton microscopy for imaging deeper in the tissue. The mammary gland may be bathed with pharmacological agents or fluorescent dyes throughout the surgery, providing a platform for acute experimental manipulations as required.

The present protocol describes a facile technique for the intravital imaging of the lactating mouse mammary gland by laser scanning confocal and multiphoton microscopy.

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory ...

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

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