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="https://cloudflare-jove-com.bdigitaluss.remotexs.co/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="https://cloudflare-jove-com.bdigitaluss.remotexs.co/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="https://cloudflare-jove-com.bdigitaluss.remotexs.co/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 San Sebastian

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. ...

Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS)

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological conditions including parasitic infection, inflammation, traumatic stress, graft-versus-host disease, diabetes and cancer1-7. In mice, MDSC express Mac-1 (CD11b) and Gr-1 (Ly6G and Ly6C) surface antigens7. It is important to note that MDSC are well studied in various tumor-bearing hosts where they are significantly expanded and suppress anti-tumor immune responses compared to na&iuml;ve counterparts7-10. However, depending on the pathological condition, there are different subpopulations of MDSC with distinct mechanisms and targets of suppression11,12. Therefore, effective methods to isolate viable MDSC populations are important in elucidating their different molecular mechanisms of suppression in vitro and in vivo.
Recently, the Ghansah group has reported the expansion of MDSC in a murine pancreatic cancer model. Our tumor-bearing MDSC display a loss of homeostasis and increased suppressive function compared to na&iuml;ve MDSC 13. MDSC percentages are significantly less in lymphoid compartments of na&iuml;ve vs. tumor-bearing mice. This is a major caveat, which often hinders accurate comparative analyses of these MDSC. Therefore, enriching Gr-1+ leukocytes from na&iuml;ve mice prior to Fluorescence Activated Cell Sorting (FACS) enhances purity, viability and significantly reduces sort time. However, enrichment of Gr-1+ leukocytes from tumor-bearing mice is optional as these are in abundance for quick FACS sorting. Therefore, in this protocol, we describe a highly efficient method of immunophenotyping MDSC and enriching Gr-1+ leukocytes from spleens of na&iuml;ve mice for sorting MDSC in a timely manner. Immunocompetent C57BL/6 mice are inoculated with murine Panc02 cells subcutaneously whereas na&iuml;ve mice receive 1XPBS. Approximately 30 days post inoculation; spleens are harvested and processed into single-cell suspensions using a cell dissociation sieve. Splenocytes are then Red Blood Cell (RBC) lysed and an aliquot of these leukocytes are stained using fluorochrome-conjugated antibodies against Mac-1 and Gr-1 to immunophenotype MDSC percentages using Flow Cytometry. In a parallel experiment, whole leukocytes from na&iuml;ve mice are stained with fluorescent-conjugated Gr-1 antibodies, incubated with PE-MicroBeads and positively selected using an automated Magnetic Activated Cell Sorting (autoMACS) Pro Separator. Next, an aliquot of Gr-1+ leukocytes are stained with Mac-1 antibodies to identify the increase in MDSC percentages using Flow Cytometry. Now, these Gr1+ enriched leukocytes are ready for FACS sorting of MDSC to be used in comparative analyses (na&iuml;ve vs. tumor- bearing) in in vivo and in vitro assays.

Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting AutoMACS

This is a rapid and comprehensive method of immunophenotyping Myeloid Derived Suppressor Cells (MDSC) and enriching Gr-1+ leukocytes from mouse spleens. This method uses flow cytometry and AutoMACS Cell Sorting to enrich for viable Gr-1+ leukocytes prior to FACS sorting of MDSC for use in vivo and in vitro assays.

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological ...

Medicine

Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The isolation of these cells offers the possibility of studying their involvement in cardiac diseases, and, in addition, provides a useful primary cell model to investigate biological mechanisms.
Here, the method for the isolation of C-MSC from arrhythmogenic cardiomyopathy patients&#39; bioptic samples is described. The endomyocardial biopsy sampling is guided in the right ventricular areas adjacent to the scar visualized by electro-anatomical mapping. The digestion of the biopsies in collagenase and their plating on a plastic dish in culture medium to allow C-MSC growth is described. The isolated cells can be expanded in culture for several passages. To confirm their mesenchymal phenotype, the description of immuno-phenotypical characterization is provided. C-MSC are able to differentiate into several cell types like adipocytes, chondrocytes, and osteoblasts: in the context of ACM, characterized by adipocyte deposits in patients&#39; hearts, the protocols for the adipogenic differentiation of C-MSC and the characterization of lipid droplet accumulation are described.

In this article, a method to isolate cardiac mesenchymal stromal cells from endomyocardial bioptic samples of arrhythmogenic cardiomyopathy patients is provided. Their characterization and the protocol to boost their adipogenic differentiation are described.

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The ...

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

Satellite Cell Isolation from Mouse Limb Muscles and Flow Cytometry Analysis

An Efficient Protocol for CUT&amp;RUN Analysis of FACS-Isolated Mouse Satellite Cells

Genome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient protocol for the fluorescence-activated cell sorting (FACS) isolation of satellite cells from the limb muscle, a tissue with a high content of structural proteins. Dissected limb muscles from adult mice were mechanically disrupted by mincing in medium supplemented with dispase and type I collagenase. Upon digestion,&#160;the homogenate was filtered through cell strainers, and cells were suspended in FACS buffer. Viability was determined with fixable viability stain, and immunostained satellite cells were isolated by FACS. Cells were lysed with Triton X-100 and released nuclei were bound to concanavalin A magnetic beads. Nucleus/bead complexes were incubated with antibodies against the transcription factor or histone modifications of interest. After washes, nucleus/bead complexes were incubated with protein A-micrococcal nuclease, and chromatin cleavage was initiated with CaCl2. After DNA extraction, libraries were generated and sequenced, and the profiles for genome-wide transcription factor binding and covalent histone modifications were obtained by bioinformatic analysis. The peaks obtained for the various histone marks showed that the binding events were specific for satellite cells. Moreover, known motif analysis unveiled that the transcription factor was bound to chromatin via its cognate response element. This protocol is therefore adapted to study gene regulation in adult mice limb muscle satellite cells.

Author Spotlight: Cistrome Analysis in Mouse Muscle Stem Cells 

Presented here is an efficient protocol for the fluorescence-activated cell sorting (FACS) isolation of mouse limb muscle satellite cells adapted to the study of transcription regulation in muscle fibers by cleavage under targets and release using nuclease (CUT&amp;RUN).

Genome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient ...

Isolation of Murine Intestinal Mesenchyme Resulting in a High Yield of Telocytes

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, fibroblasts, myofibroblasts, smooth muscle cells, immune cells, and the recently identified cell type, telocytes. Telocytes are unique mesenchymal cells with long cytoplasmic processes, reaching a distance of tens to hundreds of microns from the cell body. Telocytes have recently emerged as an important intestinal stem cell niche component, providing Wnt proteins that are essential for stem and progenitor cell proliferation.
Although protocols on how to isolate mesenchyme from the mouse intestine are available, it is not clear whether these procedures allow the efficient isolation of telocytes. Isolating telocytes efficiently requires special protocol adjustments that would allow dissociation of the strong cell-cell contact between telocytes and neighboring cells without affecting their viability. Here, available intestinal mesenchyme isolation protocols were adjusted to support the successful isolation and culture of mesenchyme containing a relatively high yield of viable single-cell telocytes.
The obtained single-cell suspension can be analyzed by several techniques, such as immunostaining, cell sorting, imaging, and mRNA experiments. This protocol yields mesenchyme with sufficiently conserved antigenic and functional properties of telocytes, and can be used for several applications. For example, they can be used for co-culture with mouse- or human-derived organoids to support organoid growth with no growth factor supplementation, to better reflect the situation in the original tissue.

Here, we present a protocol to isolate murine intestinal mesenchyme, including telocytes. These can be used for several applications, such as co-culture with mouse or human-derived organoids, to support growth and better reflect the situation in the original tissue.

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

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

Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS)

Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients

Pancreatic Tissue Dissection to Isolate Viable Single Cells

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

An Efficient Protocol for CUT&RUN Analysis of FACS-Isolated Mouse Satellite Cells

Isolation of Murine Intestinal Mesenchyme Resulting in a High Yield of Telocytes

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Isolation of Mouse Pancreatic Endothelial Cells