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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.4 &#181;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 &#215; 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 &#38; Co. KG</td>
			<td>111650</td>
			<td>Deckglaser Cover Glasses 25 mm diameter&#160;</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>&#160;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 &#956;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&#160;</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 &#38; 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.

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

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

My research delves into the immune system's role in regeneration and repair. I focus on understanding how macrophages interact with epithelial progenitor cells during salivary gland regeneration injury, and how this interaction shapes the repair process. Within the fields of immunology and regenerative medicine, recent clinical trials based at the University of Edinburgh have shown that delivery of macrophages as a therapy for liver psoriasis is effective in reducing liver scarring, and is a viable treatment for liver failure.Using and ex vivo precision cut slice culture model coupled with live imaging of fluorescent-labeled cells, allows us to explore cell-to-cell interactions in real-time, but in a state that closely resembles tissue in vivo. Understanding the localization of macrophages after injury gives us an idea of what cell types macrophages preferentially interact with after injury. This ultimately allows us to use other methods, such as single-cell RNA sequencing analysis, to try and unravel the molecular basis of such interactions to inform future therapeutic options.We will explore the bidirectional signaling between macrophages and epithelial cells and test whether saliva gland homeostasis can be rescued by exogenous replacement of these missing signals.

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

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.

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

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Research

JoVE Journal

Immunology and Infection

Collection, Embedding, Sectioning, and Staining of the Mouse Submandibular Salivary Gland for Live Cell Imaging of Cell-Cell Interactions

To begin, use forceps to remove any fat and connective tissue from the dissected submandibular salivary gland of a euthanized mouse. Then place the gland in a collection tube with ice cold HBSS solution. Next, use forceps to cut the gland into one centimeter square pieces.Heat 4%agarose to 50 degrees Celsius, and pour the solution into a 35 millimeter dish. Pour a small amount of the agarose solution into a separate dish and add the gland pieces into it. Then swirl the pieces in the excess agarose to coat it.Next place four to six pieces of the gland flat in the first dish with agarose. Place the lid on the dish and transfer it to an ice box, covering the plate with ice to cool and set. To section the gland pieces, first, use a scalpel to carefully cut around the gland embedded agarose block.Next, apply a drop of super glue to the agarose block and attach it to the stage of a vibratome. Now fill the vibratome chamber with ice cold PBS containing 1%penicillin streptomycin. With a scalpel, trim off any excess agarose and create five millimeter gaps between each piece of gland.Align the vibratome blade with the agarose block and set the start and end points of the sections. Cut the tissue block into 150 micrometer thick sections at a low speed and high vibration. Once the sections have been cut, use a paintbrush to pick up the slices and place them in a dish with prewarmed RPMI media containing the antibiotics.To culture the submandibular slices, first, add 1.5 milliliters of RPMI media to the wells of a six well plate. Place 0.4 micrometer filters into the wells. Next, with the help of a paintbrush, carefully transfer one to six slices onto each filter.Then incubate the plate at 37 degrees Celsius under 5%carbon dioxide. After irradiating some experimental plates with gamma radiation to induce injury, return the plates to the incubator. Next, fill the wells of a 24 well plate with 500 microliters of culture media.With a paintbrush, lift the slices from the filter and gently submerge them in the wells. Incubate the slices in the relevant nuclear stains. Then incubate the slices with antibodies for two hours at 37 degrees Celsius under gentle agitation.Submerge the slices in culture media three times to wash them. Let the slices incubate in each wash solution for 10 minutes at room temperature under gentle agitation. Next, use forceps to remove the tape from a double-sided imaging spacer.Stick the spacer to the bottom of a glass-bottomed six well plate, then pipette 50 microliters of media into the gap in the center of the spacer. Place the slice into the media ensuring it lays flat. Then with a pipette, carefully remove 20 microliters of the media from the gap.Use forceps to carefully remove the tape from the top side of the spacer and place a 25 millimeter circular cover slip over it. Press around the spacer edges to ensure firm attachment of the cover slip. Image the slice on a confocal microscope.Non-irradiated slices of the submandibular gland cultured for seven days retained their MT signal and epithelial architecture. However, at three days post-irradiation, acinar and ductal cell atrophy was observed. Caspase positive cells were seen at four days post-irradiation.Elevated gamma H2AX was observed in irradiated slices, indicative of in vivo DNA damage. Realtime imaging of macrophages confirmed phagocytosis of epithelial cells in the slice culture model. Individual cells can be detected and segmented for subsequent analysis of cell behavior, such as migration.

816 Views.  The University of Edinburgh.  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.

Real-Time Live Imaging of Mouse Submandibular Gland Regeneration

Summary