The authors thank Adi Sasson for the technical assistance and Helen Guez for the critical reading of the manuscript. This work was supported by European Research Council starting grant no. 336204.

Experiments were conducted according to protocols approved by the Committee on Animal Research at Tel Aviv University.
1. Isolation of Pancreatic Tissue from Mice
<ol>
	<li>For adult mice:
	<ol>
		<li>Prepare the digestion buffer: 0.4 mg/mL collagenase P and 0.1 ng/mL DNase I dissolved in Hanks&#39; Balanced Salt Solution (HBSS). Freshly prepare 5 mL of buffer per mouse in 15-mL conical tubes and keep them on ice until use.</li>
		<li>Euthanize the mice according to institutional guideline.</li>
		<li>Dissect each mouse to remove the pancreas (for instruction, please see Reference 18); place it in a culture dish containing HBSS. It is possible to perform this stage under a stereomicroscope, and to increase digestion efficiency, cut the tissue into 2 - 4 pieces.</li>
		<li>Place the pancreatic tissue into a tube containing digestive buffer (prepared in step 1.1.1). Keep the tubes on ice until all mice are dissected and pancreatic tissues collected.</li>
		<li>Repeat steps 1.1.2 - 1.1.4 with the remaining mice.</li>
	</ol>
	</li>
	<li>For embryos and neonatal mice:
	<ol>
		<li>Prepare the digestion buffer: 0.4 mg/mL collagenase P and 0.1 ng/mL DNase I dissolved in HBSS. For embryos and pups at postnatal day 7 (p7) or younger, freshly prepare 1.5 - 2 mL of buffer per mouse in a 15-mL conical tube. For p7 or older pups, prepare 3 - 4 mL of buffer per mouse in a 15-mL conical tube. Keep on ice until use.</li>
		<li>Euthanize the mice according to institutional guideline. For embryos, remove all embryos from their mother&#39;s uterus and place them in a 10-mm culture dish containing PBS. Dissect one embryo at a time.</li>
		<li>Lay the mouse on its back, with its head pointing away from the investigator. Using fine forceps, pull up the abdominal skin and open at the midline to expose the abdominal cavity up to the mouse diaphragm18.</li>
		<li>Using fine forceps, detach the liver from the back abdominal wall. With a continuous motion toward the mouse tail, scoop out the internal organs (including the liver, stomach, spleen, intestines, kidney, and pancreas) and place them in a culture dish containing HBSS.</li>
		<li>Under a stereomicroscope, remove the liver and kidneys to expose the pancreas (Figure 1). Using fine forceps, detach the pancreas from the stomach, duodenum, and finally, from the spleen.</li>
		<li>Place the pancreatic tissue into a tube containing digestive buffer (prepared in step 1.2.1). For p14 or older pups, cut the tissue into 2 pieces to increase the digestion efficiency. Keep the tubes on ice until all mice are dissected and pancreatic tissues collected.</li>
		<li>Repeat steps 1.2.2 - 1.2.6 with the remaining mice.</li>
	</ol>
	</li>
</ol>
2. Pancreas Digestion
<ol>
	<li>Incubate the tubes containing the pancreatic tissue (in digestion buffer) in a heating block at 37 &#176;C for 30 min with agitation at 700 rpm. After 15 min, manually shake the tubes by inverting them 3 - 4 times and place them back in the heating block. 
	NOTE: If using a heating block without agitation capabilities, invert the tubes every 5 - 10 min. Make sure that the tissue is properly digested. If not, cut the tissue into smaller pieces before incubation or increase the agitation.</li>
	<li>To stop the tissue digestion, place the tubes on ice and add 10 mL of cold HBSS to each tube. Centrifuge the tubes at 4 &#176;C and 300 x g for 5 min and aspirate the supernatant.</li>
	<li>Re-suspension:
	<ol>
		<li>For adult mice and p14 or older pups, re-suspend the pellet with 6 mL of PBS and strain it through a 70-&#181;m cell strainer placed on top of a new 15-mL conical collection polypropylene tube. To ensure maximal cell recovery, wash the original tube with an additional 6 mL of PBS and strain it through the cell strainer onto the collection tube.</li>
		<li>For embryos and pups younger than p14, re-suspend the pellet with 2 mL of PBS and strain it through a 35-&#181;m cell strainer placed on top of a 5-mL round-bottom polystyrene collection tube (FACS tube). To ensure maximal cell recovery, wash the original tube with an additional 2 mL of PBS and strain it through the cell strainer onto the collection tube.</li>
	</ol>
	</li>
	<li>Centrifuge the tubes at 4 &#176;C and 300 x g for 5 min and aspirate the supernatant. Remove the liquid carefully, as cells are loosely attached to the polystyrene tube.</li>
</ol>
3. Preparing Labeled Single Cells
<ol>
	<li>Buffer preparations: For cell isolation by FACS, prepare sorting buffer by mixing PBS (without calcium chloride and magnesium chloride), 5% fetal bovine serum (FBS), and 5 mM EDTA. For flow cytometry analysis, prepare analysis buffer by supplementing sorting buffer with 0.05% sodium azide. Both buffers may be stored at 4-8 &#176;C for up to 2 months.</li>
	<li>Re-suspend cells in sorting or analysis buffer. Re-suspend each pancreas isolated from embryos and pups younger than p7 in 1 mL, from pups between p8 and one month old in 1.5 mL, and from mice older than one month in 3 mL. Strain the cells through a 35-&#181;m cell strainer placed on top of a round-bottom polystyrene tube (FACS tube).</li>
	<li>For staining controls, take out 50- to 100-&#181;L aliquots from the samples made in the previous step (not more than 5% of the total sample volume) and place them into new FACS tubes; include a tube for each fluorophore used, including DAPI and fluorescent proteins, as well as for an unstained control. 
	NOTE: When using transgenic mice with cells expressing a fluorescent protein, or when cell number is limited, include a non-transgenic tissue as a staining control. If cells expressing a fluorescent protein are used without further staining, proceed to step 3.8.</li>
	<li>Spin-down the cells at 300 x g for 5 min at 4 &#176;C and aspirate the supernatant.</li>
	<li>For blocking, re-suspend the cells with blocking solution (100 &#181;L sorting or analysis buffer supplemented with 1 &#181;L of goat IgG) and incubate for 30 min on ice.</li>
	<li>To stain cell surface markers, prepare a mixture that includes all desired fluorophore-conjugated antibodies in a volume of 100 &#181;L per sample . Prepare 2x antibody dilutions in sorting or analysis buffer (i.e., dilute the antibody to obtain double the required concentration; if a final dilution of 1:200 is desired, dilute the antibody 1:100). Without washing the blocking solution, add 100 &#181;L of the mixture to the sample cells to achieve a final staining volume of 200 &#181;L. Incubate for 30-60 min on ice and in the dark.</li>
	<li>To set the analysis or sorting parameters (see steps 4.3 and 5.2), prepare additional tubes that contain only one of the antibodies used (staining control). Prepare 2x antibody dilutions in sorting or analysis buffer (as described in step 3.6). Make sure to include a single staining control dilution for each fluorophore used, as well as an unstained control. Without washing the blocking solution, add 100 &#181;L of antibody mixes to the cells (prepared in step 3.3) to achieve a final staining volume of 200 &#181;L. Incubate for 30 - 60 min on ice and in the dark.</li>
	<li>Wash by filling each tube with analysis or sorting buffer to a maximal volume of 4 mL. Optionally, re-strain the cells through a 35-&#181;m cell strainer (as described in step 3.2).</li>
	<li>Centrifuge at 300 x g for 5 min at 4 &#176;C and remove the supernatant carefully.</li>
	<li>Re-suspend the samples in analysis or sorting buffer. Re-suspend the cells isolated from embryos in 500 &#181;L, from pups younger than one month in 1 mL, from 1 - 3 months old in 2 mL, and from mice older than 3 months in 3 mL. Staining controls may be re-suspended in 300 &#181;L of buffer.</li>
	<li>Add 200 ng/mL of DAPI to re-suspended cells to identify the dead cells. Make sure to include a tube containing unstained cells without DAPI for cytometer settings. Proceed to cell sorting (step 4) or analysis (step 5).</li>
</ol>
4. Cell Sorting
<ol>
	<li>Preparations:
	<ol>
		<li>For cell sorting prior to RNA extraction, coat a 1.5-mL collecting tube with RNase inhibitor immediately before sorting. To this end, add 1 mL of sorting buffer, containing 0.01 U/mL RNase inhibitor, to a sterile 1.5-mL collection tube. After 5 min, vortex the tube and remove the liquid.</li>
		<li>For cell sorting prior to cell culture, add 3 mL of sterile culturing media (Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 20% FBS, 1% L-glutamine, and 1% penicillin-streptomycin) into a sterile 15-mL collection tube.</li>
	</ol>
	</li>
	<li>Before loading a tube into a FACS sorter, vortex it briefly to re-suspend the cells. Keep the remaining tubes on ice.</li>
	<li>Start by analyzing the staining controls to determine the sorting parameters (e.g., voltage and compensation) and sorting gates (e.g., total cell population, live DAPI-negative cells, and cell populations to be sorted).</li>
	<li>Once the sorting parameters and gates are set up, load the samples and initiate cell sorting into the collection tubes. 
	NOTE: Sorting conditions are highly dependent on the instrument. We use a nozzle width of 100 &#181;m, a pressure of 23.1 psi, and a maximal sorting speed of 5.</li>
	<li>Proceed to RNA extraction or the culturing of sorted cells. 
	NOTE: For RNA extraction, centrifuge the cells at 2,000 x g for 5 min and remove the excess liquid before continuing with a standard extraction protocol. For culturing cells, if the cells were sorted under non-sterile conditions, wash them twice by filling the tube with culturing medium and centrifuging it at 300 x g for 7 min before culturing in order to minimize their contamination.</li>
</ol>
5. Cell Analysis by Flow Cytometry
<ol>
	<li>Before loading each tube into the cytometer, vortex it briefly to re-suspend the cells. Keep the remaining tubes on ice.</li>
	<li>Start by analyzing the unstained and single-stained samples in order to determine the analysis parameters (e.g., voltage and compensation).</li>
	<li>Once the analysis parameters are set up, load each sample, including the staining control, and record the results. Analyze the obtained results using flow cytometry analysis software.</li>
</ol>

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The authors have nothing to disclose.

Here, we describe a method to isolate and analyze cells of the pancreatic microenvironment. This method can be used to isolate mesenchymal cells from embryonic and adult pancreatic tissue. In addition, we successfully used this protocol to isolate endothelial cells from the adult and neonatal pancreas5,17. However, it may not be suitable for obtaining a reproducible single-cell suspension of pancreatic epithelial cells (alternative protocols are described in References 18, 23, and 24). Using this method, fluorescently-labeled cells, either expressing fluorescent proteins or immunostained for surface markers, can be purified by FACS or analyzed by flow cytometry. RNA can be extracted from purified cells to profile their gene expression pattern. Alternatively, purified cells can be cultured to establish a cell line for subsequent proteomic analysis. This method will enable the characterization of factors expressed by the pancreas microenvironment, which govern its organogenesis, physiology, and pathophysiology.
The pancreatic mesenchyme supports tissue organogenesis by promoting the proliferation of precursors and differentiated cells5,9. These cells were shown to support the expansion of human embryonic stem cell (hESC)-derived pancreatic progenitors17,25,26. Therefore, delineating the identity of embryonic mesenchymal factors would facilitate current efforts to generate insulin-producing beta cells from hESCs and induced pluripotent stem cells (iPSCs) as a potential cure to diabetes. Mouse genetic studies allowed the identification of growth factors, such as Fgf10, that are produced by the mesenchyme to promote pancreatic epithelium expansion during the early stages of pancreas development3,9. With the aim of identifying additional factors expressed in the embryonic mesenchyme, we isolated these cells using laser-captured microdissection, extracted their RNA, and performed gene expression analysis26. However, in addition to being labor-intense, this method relies on identifying cells based on their morphological features, which restricts its use to developmental stages prior to the branching of the epithelium into the surrounding mesenchyme (i.e., e12.5). To characterize mesenchymal cells at later developmental stages, we employed the method described here5,17.
We used this method to analyze surface marker expression by neonatal pancreatic mesenchyme5. In addition, mesenchymal cells were isolated from embryonic and neonatal pancreatic tissue of Nkx3.2-Cre;R26-EYFP mice, based on their fluorescent labeling in this mouse line, and were cultured to establish cell lines17. The proteomic analysis of these cells allowed for the identification of factors secreted by the pancreatic mesenchyme with the ability to promote hESC-derived pancreatic progenitors17. We further used this cell isolation method to purify mesenchymal cells from adult pancreatic tissues for RNA extraction and gene expression analysis17. Therefore, this method can be used to identify genes and proteins expressed by the pancreatic mesenchyme, with the ability to support pancreatic cell development.
Pancreatic mesenchymal cells were further shown to play a role in pancreas tumorigenesis. PDAC is characterized by the formation of a fibroblast-rich desmoplastic stroma comprised of fibroblasts, immune cells, and ECM27. While the stroma was thought to promote the development of many types of cancer, it was shown to restrain PDAC progression15,16,28. This suggests that components of the pancreatic stroma secrete factors that inhibit tumorigenesis. Furthermore, changes in stroma cellular composition as well as in cell phenotype can underlie their effect on epithelial cells15,16,28. The method described here can therefore assist in characterizing the different cell types that make up a PDAC stroma as compared to healthy pancreatic tissue. It would further allow the purification of the different stromal cell types to characterize potential changes in their gene expression profiles during PDAC progression. However, due to changes in pancreatic ECM composition during tumorigenesis27, adjustments of the tissue digestion parameters, such as the inclusion of additional collagenase types or increasing the incubation time, may be required.

Energy homeostasis and food digestion in mammals depend on proper pancreatic function. The adult pancreas is comprised of two main cellular compartments: the exocrine and the endocrine. Exocrine cells, including the acinar cells that produce and secrete digestive enzymes and the duct cells that transport these enzymes to the gut, encompass more than 80% of the total pancreatic mass1. Endocrine cells, which include insulin-producing beta cells and glucagon-producing alpha cells, are organized in the islets of Langerhans that are embedded in the exocrine tissue and secrete hormones to regulate blood glucose levels2.
Pancreatic cells acquire their differentiated fate through a highly-regulated, multistep process3. Evidence suggests that extrinsic cues provided by neuronal, endothelial, and mesenchymal cells guide pancreatic cell differentiation and proliferation in the embryo3,4,5. One example is the requirement of the aorta for the specification of early pancreatic precursors6. Later in development, endothelial cells were shown to play a central role in the development of both pancreatic endocrine and exocrine cells and to promote beta-cell differentiation4,6,7. Mesenchymal cells were shown to support the survival and expansion of common pancreatic progenitors, mainly through the secretion of the growth factor Fgf108,9. We have further shown that these cells support the proliferation of endocrine and exocrine precursors, as well as of differentiated cells (including acinar and beta cells) in the embryonic pancreas5. Recently, mesenchymal cells were further shown to regulate endocrine cells differentiation10.
In the adult, beta-cell function and mass were shown to depend on cells in their microenvironment, including neuronal, immune, and endothelial cells, as well as pericytes11,12,13. During injury, endothelial cells were shown to recruit immune cells to the pancreas to promote beta-cell replication13. Endothelial cells were further shown to produce extracellular matrix (ECM) components to support insulin expression and beta-cell function14. We recently demonstrated the requirement of islet pericytes for beta-cell function11. Lastly, cells of the pancreatic stroma were shown to regulate the progression of pancreatic ductal adenocarcinoma (PDAC)15,16. However, the identity of extrinsic cues that guide pancreas development, function, and tumorigenesis are largely unknown.
Identifying cues provided by cells of the pancreas microenvironment requires characterizing the genes and proteins expressed by these cells. This depends on isolating these cells from the pancreas in order to perform gene expression and proteomic analyses and/or on establishing cell lines. Here, we propose a method to isolate mesenchymal cells of the pancreas microenvironment by utilizing tissue enzymatic digestion and fluorescence-activated cell sorting (FACS) of either immunofluorescently-labeled cells or cells expressing fluorescent proteins. This protocol was successfully performed to isolate and analyze yellow fluorescent protein (YFP)-expressing mesenchymal cells of the embryonic, neonatal, and adult pancreas5,17.

<table><tbody><tr><td>Collagenase P</td><td>Roche</td><td>11213865001</td><td></td></tr><tr><td>DNase</td><td>Sigma-Aldrich</td><td>D5025-15KU</td><td>The effective units should be at least 2,000 unitz/mL protein</td></tr><tr><td>Hanks&amp;rsquo; Balanced Salt solution (HBSS)</td><td>Sigma-Aldrich</td><td>H6648</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Biological Industries</td><td>04-127-1A</td><td></td></tr><tr><td>EDTA</td><td>Biological Industries</td><td>01-862-1A</td><td></td></tr><tr><td>Sodium Azide</td><td>Sigma-Aldrich</td><td>S2002</td><td></td></tr><tr><td>Goat IgG serum</td><td>Sigma-Aldrich</td><td>G9023</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542</td><td></td></tr><tr><td>RNAse inhibitor</td><td>Invitrogen</td><td>N8080119</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Invitrogen</td><td>11965092</td><td></td></tr><tr><td>L-Glutamine</td><td>Biological Industries</td><td>03-020-1B</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Biological Industries</td><td>03-031-1B</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline (PBS)</td><td>Sigma-Aldrich</td><td>D8537</td><td>Without Calcium Chloride and Magnesium Chloride</td></tr><tr><td>1.5 mL tubes</td><td>Sarstedt</td><td>72 690</td><td></td></tr><tr><td>15 mL conical tube</td><td>Corning</td><td>430052</td><td></td></tr><tr><td>Round Bottom Polystyrene 5 mL tube</td><td>Corning</td><td>352008</td><td>FACS tube. Make sure tube is compatible with the flow cytomter to be used, as there are slight differences in required tubes between brands</td></tr><tr><td>5 mL tube with 35 &amp;mu;m cell strainer Snap Cap</td><td>Corning</td><td>352235</td><td>FACS tube</td></tr><tr><td>70 &amp;mu;m cell strainer&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-098-462</td><td></td></tr><tr><td>Heating block with agitation&amp;nbsp;</td><td>Eppendorf</td><td>ThermoMixer C</td><td></td></tr><tr><td>Centrifuge</td><td>ThermoFisher</td><td>Heraeus Megafuge 40R</td><td></td></tr><tr><td>Steromicroscope</td><td>Nikon</td><td>SMZ 745</td><td></td></tr><tr><td>Cell sorter</td><td>BD Biosciences</td><td>FACSAria IIu</td><td></td></tr><tr><td>flow cytometer</td><td>Beckman Coulter</td><td>Gallios&amp;nbsp;</td><td></td></tr></tbody></table>

isolating and analyzing cells of the pancreas mesenchyme by flow cytometry

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, including endothelial, neuronal, and mesenchymal cells were shown to regulate cell differentiation and proliferation in the embryonic pancreas. In the adult, the function and mass of insulin-producing cells were shown to depend on cells in their microenvironment, including pericyte, immune, endothelial, and neuronal cells. Lastly, changes in the pancreas microenvironment were shown to regulate pancreas tumorigenesis. However, the cues underlying these processes are not fully defined. Therefore, characterizing the different cell types that comprise the pancreas microenvironment and profiling their gene expression are crucial to delineate the tissue development and function under normal and diseased states. Here, we describe a method that allows for the isolation of mesenchymal cells from the pancreas of embryonic, neonatal, and adult mice. This method utilizes the enzymatic digestion of mouse pancreatic tissue and the subsequent fluorescence-activated cell sorting (FACS) or flow-cytometric analysis of labeled cells. Cells can be labeled by either immunostaining for surface markers or by the expression of fluorescent proteins. Cell isolation can facilitate the characterization of genes and proteins expressed in cells of the pancreas mesenchyme. This protocol was successful in isolating and culturing highly enriched mesenchymal cell populations from the embryonic, neonatal, and adult mouse pancreas.

The pancreatic mesenchyme is required during development and adulthood. The method described here allows the isolation of mesenchymal cells from the embryonic, neonatal, and adult pancreas. Mesenchymal cells, but no other cell types, express yellow fluorescent protein (YFP) in the pancreas of Nkx3.2-Cre;R26R-YFP mice5,11,17,19. During development, Nkx3.2 (also known as BapX1) is expressed by embryonic pancreatic, stomach, and gut mesenchyme, as well as in a subset of skeletal somites19,20,21. This gene was expressed in the pancreatic mesenchyme from e9.5 until e11.5, allowing gene expression under the control of Nkx3.2-Cre from e9.55,19,20. Based on this labeling, cells can be purified from bulk pancreatic tissue using flow cytometry. Figure 2 shows a flow-cytometry analysis of single cells from embryonic, neonatal, and adult pancreatic tissues, isolated as described here. Whereas non-transgenic pancreatic tissues did not contain fluorescent cells, Nkx3.2-Cre;R26R-YFP pancreatic tissue from all analyzed ages contains a distinct YFP-labeled cell population (Figure 2; marked with gates).
Following the method described here, cells expressing fluorescent proteins can be either purified or analyzed by flow cytometry, with or without additional immunostaining. For example, after sorting based on fluorescent labeling, mesenchymal cells can be cultured to establish a cell line (for at least five passages), as shown in Figure 3A. Note the fibrocytic morphology of the cultured cells, typical to mesenchymal cells. In addition, this system was used to analyze gene expression by sorted cells. To this end, RNA was extracted from sorted mesenchymal cells to synthesize cDNA, and gene expression levels were analyzed by qPCR. Such analysis revealed that sorted cells express the pan-mesenchymal marker vimentin (Figure 3B). Lastly, surface marker expression by pancreatic cells can be analyzed by flow-cytometry. For example, we isolated cells from the pancreatic tissue of Nkx3.2-Cre;R26R-YFP adult mice using the method described here and stained them with the cell surface glycoprotein CD9, which was reported to be expressed by fibroblasts22. As shown in Figure 3C, all fluorescently-labeled cells in the Nkx3.2-Cre;R26R-YFP pancreas express CD9.
<img alt="Figure 1" src="/files/ftp_upload/55344/55344fig1.jpg" /> 
Figure 1: Embryonic and neonatal pancreatic tissue. Isolated whole gastrointestinal tract, including the stomach, spleen, intestine, and pancreas, of an e15.5 embryo (A) and a p4 pup (B). The pancreatic tissue is demarcated with a blue line. <a href="http://cloudflare.jove.com/files/ftp_upload/55344/55344fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55344/55344fig2.jpg" /> 
Figure 2: Mesenchymal cells are fluorescently labeled in Nkx3.2-Cre;R26R-YFP pancreatic tissue. Flow-cytometry analysis of pancreatic cells isolated from non-transgenic (left panels) and Nkx3.2-Cre;R26R-YFP transgenic (right panels) mice at various ages: embryonic (A), neonatal (B), and adult (C). Cells were analyzed for side scatter (y-axis) and yellow fluorescence (x-axis). Gates (marked with &#34;D&#34;) indicated the presence of a YFP+ cell population in transgenic mice but not in non-transgenic controls. <a href="http://cloudflare.jove.com/files/ftp_upload/55344/55344fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55344/55344fig3.jpg" /> 
Figure 3: Analyses of isolated pancreatic cells. (A) Cells sorted from the pancreatic tissue of Nkx3.2-Cre;R26R-YFP neonatal mice (as described in Figure 2B) were cultured to establish a cell line. Cultured cells were imaged for fluorescence (green; right and left panels) and phase contrast (gray; right panel). (B) Bar diagram showing Vimentin1 (Vim1) expression levels by YFP+-sorted cells from the pancreatic tissue of Nkx3.2-Cre;R26R-YFP adult mice (as described in Figure 1C; green) as compared to unsorted pancreatic tissue (black). RNA was extracted and gene expression was analyzed by qPCR; expression was normalized to Cyclophilin. N = 4. ***P &#60; 0.001. Data represent the mean &#177; SD. (C) Flow-cytometry analysis of dispersed pancreatic cells from Nkx3.2-Cre;R26R-YFP adult mice immuno-stained with allophycocyanin (APC)-conjugated anti-CD9 antibody and analyzed for APC (y-axis) and yellow fluorescence (x-axis). <a href="http://cloudflare.jove.com/files/ftp_upload/55344/55344fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry at JoVE.com

Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry

Here, we describe a method for the isolation of cells in the pancreas microenvironment from embryonic, neonatal and adult mouse tissue, focusing on the isolation of mesenchymal cells. This method allows profiling of cell gene expression and protein secretion in order to elucidate the extrinsic signals that regulate pancreas development, function, and tumorigenesis.

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

12.1K Views.  Sackler Faculty of Medicine, Tel Aviv University. Here, we describe a method for the isolation of cells in the pancreas microenvironment from embryonic, neonatal and adult mouse tissue, focusing on the isolation of mesenchymal cells. This method allows profiling of cell gene expression and protein secretion in order to elucidate the extrinsic signals that regulate pancreas development, function, and tumorigenesis.

Video: Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

Cancer Research

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Pancreatic Tissue Dissection to Isolate Viable Single Cells

The pancreas includes two major systems: the endocrine system, which produces and secretes hormones, and the exocrine system, which accounts for approximately 90% of the pancreas and includes cells that produce and secrete digestive enzymes. The digestive enzymes are produced in the pancreatic acinar cells, stored in vesicles called zymogens, and are then released into the duodenum via the pancreatic duct to initiate metabolic processes. The enzymes produced by the acinar cells can kill cells or degrade cell-free RNA. In addition, acinar cells are fragile, and common dissociation protocols result in a large number of dead cells and cell-free proteases and RNases. Therefore, one of the biggest challenges in pancreatic tissue digestion is recovering intact and viable cells, especially acinar cells. The protocol presented in this article shows a two-step method that we developed to meet this need. The protocol can be used to digest normal pancreata, pancreata that include pre-malignant lesions, or pancreatic tumors that include a large number of stromal and immune cells.

Pancreatic metaplastic cells are precursors of malignant cells that give rise to pancreatic tumors. However, isolating intact viable pancreatic cells is challenging. Here, we present an efficient method for pancreatic tissue dissociation. The cells can then be used for single-cell RNA sequencing (scRNA-seq) or for two- or three-dimensional co-culturing.

The pancreas includes two major systems: the endocrine system, which produces and secretes hormones, and the exocrine system, which accounts for ...

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

Biology

Adoptive Therapy Using Tumor-Infiltrating Lymphocytes in Pancreatic Cancer Models

Flow Cytometry-Based Isolation and Therapeutic Evaluation of Tumor-Infiltrating Lymphocytes in a Mouse Model of Pancreatic Cancer

Pancreatic cancer is an aggressive malignancy with a dismal prognosis and limited therapeutic options. Adoptive cell therapy, which involves isolating and activating a patient&#39;s own immune cells, such as tumor-infiltrating lymphocytes (TILs), before re-infusing them, represents a promising experimental approach. However, techniques for adoptive cell transfer in preclinical pancreatic cancer models are not well established. Here, we describe a detailed protocol for adoptive cell therapy using TILs from a syngeneic pancreatic cancer mouse model. The procedure involves implanting live or irradiated mouse pancreatic cancer cells in fluorescence-labeled reporter mice to initiate immune cell influx, then isolating lymphocytes from primary tumors via flow cytometry sorting and/or activating and expanding tumor-reactive T cells ex vivo, and adoptively transferring these activated T cells intraperitoneally into tumor-bearing mice, followed by interleukin-2 administration.&#160;Bioluminescent tumor imaging allows for longitudinal monitoring of orthotopic tumor growth and response to therapy, especially evaluating the tumor-specific cytotoxic effects. This approach recapitulates the logistics involved in developing adoptive cell transfer therapies for pancreatic cancer patients. The results demonstrate enhanced antitumor efficacy of adoptively transferred tumor-reactive T cells compared to irrelevant lymphocyte controls. This versatile methodology enables the in vivo study of adoptive immunotherapy in pancreatic cancer as well as the optimization of cell processing parameters and combination treatment regimens.

We describe a method to utilize tumor-infiltrating lymphocytes (TILs) from mice through flow cytometry for adoptive cell transfer. This protocol aims to verify the specific cytotoxicity of TILs against tumors in a syngeneic pancreatic cancer mouse model, providing insights into the development of adoptive cell therapies for pancreatic cancer.

Pancreatic cancer is an aggressive malignancy with a dismal prognosis and limited therapeutic options. Adoptive cell therapy, which involves isolating and ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Pancreatic Islet Embedding for Paraffin Sections

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a mixed cell population in a structured architecture that impacts function, and human islets are widely variable in cell type composition. Current frequently used methods to study cultured islets include molecular studies performed on whole islets, lumping disparate islet cell types together, or microscopy or molecular studies on dispersed islet cells, disrupting islet architecture. For in vivo islet studies, paraffin-embedded pancreas sectioning is a powerful technique to assess cell-specific outcomes in the native pancreatic environment. Studying post-culture islets by paraffin sectioning would offer several advantages: detection of multiple outcomes on the same islets (potentially even the exact-same islets, using serial sections), cell-type-specific measurements, and maintaining native islet cell-cell and cell-substratum interactions both during experimental exposure and for analysis. However, existing techniques for embedding isolated islets post-culture are inefficient, time consuming, prone to loss of material, and generally produce sections with inadequate islet numbers to be useful for quantifying outcomes. Clinical pathology laboratory cell block preparation facilities are inaccessible and impractical for basic research laboratories. We have developed an improved, simplified bench-top method that generates sections with robust yield and distribution of islets. Fixed islets are resuspended in warm histological agarose gel and pipetted into a flat disc on a standard glass slide, such that the islets are distributed in a plane. After standard dehydration and embedding, multiple (10+) 4 - 5 &#181;m sections can be cut from the same islet block. Using this method, histological&#160;and immunofluorescent analyses can be performed on mouse, rat, and human islets. This is an effective, inexpensive, time-saving approach to assess cell-type-specific, intact-architecture outcomes from cultured islets.

Ex vivo pancreatic islet studies are important for diabetes research. Existing techniques to study cultured islets in their native 3-dimensional architecture are time consuming, inefficient, and infrequently used. This work describes a new, simple, and efficient method for generating high-quality paraffin sections of whole cultured islets.

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a ...

Quantitative Analysis of Mitophagy in Murine Pancreatic &#946;-Cells

Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic &#946;-Cells

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient insulin release. Advanced quantitative assessments of mitophagy often require the use of genetic reporters. The mt-Keima mouse model, which expresses a mitochondria-targeted pH-sensitive dual-excitation ratiometric probe for quantifying mitophagy via flow cytometry, has been optimized in &#946;-cells. The ratio of acidic-to-neutral mt-Keima wavelength emissions can be used to robustly quantify mitophagy. However, using genetic mitophagy reporters can be challenging when working with complex genetic mouse models or difficult-to-transfect cells, such as primary human islets. This protocol describes a novel complementary dye-based method to quantify &#946;-cell mitophagy in primary islets using MtPhagy. MtPhagy is a pH-sensitive, cell-permeable dye that accumulates in the mitochondria and increases its fluorescence intensity when mitochondria are in low pH environments, such as lysosomes during mitophagy. By combining the MtPhagy dye with Fluozin-3-AM, a Zn2+ indicator that selects for &#946;-cells, and Tetramethylrhodamine, ethyl ester (TMRE) to assess mitochondrial membrane potential, mitophagy flux can be quantified specifically in &#946;-cells via flow cytometry. These two approaches are highly complementary, allowing for flexibility and precision in assessing mitochondrial quality control in numerous &#946;-cell models.

Author Spotlight: Assessment of Mitophagy Flux in Pancreatic &#946;-Cells Using Effective and Robust Complementary Approaches 

This protocol outlines two methods for the quantitative analysis of mitophagy in pancreatic &#946;-cells: first, a combination of cell-permeable mitochondria-specific dyes, and second, a genetically encoded mitophagy reporter. These two techniques are complementary and can be deployed based on specific needs, allowing for flexibility and precision in quantitatively addressing mitochondrial quality control.

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient ...

Streamlining Mouse Pancreatic Endothelial Cell Extraction for Disease Studies

Isolation of Mouse Pancreatic Endothelial Cells

The pancreas is a vital organ for maintaining metabolic balance within the body, in part due to its production of metabolic hormones such as insulin and glucagon, as well as digestive enzymes. The pancreas is also a highly vascularized organ, a feature facilitated by the intricate network of pancreatic capillaries. This extensive capillary network is made up of highly fenestrated endothelial cells (ECs) important for pancreas development and function. Accordingly, the dysfunction of ECs can contribute to that of the pancreas in diseases like diabetes and cancer. Thus, researching the function of pancreatic ECs (pECs) is important not only for understanding pancreas biology but also for developing its pathologies. Mouse models are valuable tools to study metabolic and cardiovascular diseases. However, there has not been an established protocol with sufficient details described for the isolation of mouse pECs due to the relatively small population of ECs and the abundant digestive enzymes potentially released from the acinar tissue that can lead to cell damage and, thus, low yield. To address these challenges, we devised a protocol to enrich and recover mouse pECs, combining gentle physical and chemical dissociation and antibody-mediated selection. The protocol presented here provides a robust method to extract intact and viable ECs from the whole mouse pancreas. This protocol is suitable for multiple downstream assays and may be applied to various mouse models.

This protocol describes the isolation of mouse endothelial cells from whole pancreas.

The pancreas is a vital organ for maintaining metabolic balance within the body, in part due to its production of metabolic hormones such as insulin and ...

Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry

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Rozdziały w tym wideo

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic Tissue Dissection to Isolate Viable Single Cells

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Flow Cytometry-Based Isolation and Therapeutic Evaluation of Tumor-Infiltrating Lymphocytes in a Mouse Model of Pancreatic Cancer

A System for ex vivo Culturing of Embryonic Pancreas

Pancreatic Islet Embedding for Paraffin Sections

Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic β-Cells

Isolation of Mouse Pancreatic Endothelial Cells