In loving memory of Rich West, a brilliant scientist, colleague, and dear friend.&#160;We would like to thank the University of Delaware Flow Cytometry and BioImaging Core as part of the Delaware Biotechnology Institute for their ongoing contributions to our studies. We would also like to thank Emma Hudgins for the careful review and editing of the manuscript. Our work is supported by the National Institute of General Medical Sciences P20GM113125-6564 (I.S. Fancher). This project was also supported by the Delaware INBRE program, with a grant from the NIGMS (P20GM103446) from the National Institutes of Health and the State of Delaware (I.S. Fancher), and a University of Delaware General University Research grant (I.S. Fancher). This content is solely the responsibility of the authors and does not necessarily represent the official views of NIH.

The use of animals in these studies was approved by the University of Delaware, Institutional Animal Care and Use Committee (#1372).
1. Tissue dissection and cleaning
NOTE: 10- to 12-week-old C57BL/6J mice are used in the video protocol. Please refer to Figure 1 for the schematic and the desired outcome of tissue dissection and cleaning arteries and refer to the Table of Materials for the list of supplies and manufacturer information.
<ol>
	<li>Euthanize the mouse by CO2 asphyxiation followed by cervical dislocation.</li>
	<li>Secure the mouse using dissection pins and spray the ventral surface with 70% ethanol.</li>
	<li>Isolation of subcutaneous adipose tissues located by each hindlimb 
	&#8203;NOTE: Dissecting tools needed: straight Bonn scissors (9 cm) and blunt, curved Graefe forceps.
	<ol>
		<li>Use forceps to lift the skin right above the genitals and make a small (2 mm) incision in the skin.</li>
		<li>Insert the tip of the scissors into the incision site and make a 4-5 cm midline incision, beginning at the initial incision and dissecting cranially to the base of the sternum. Be cautious to not penetrate the abdominal cavity.</li>
		<li>Make a 1 cm lateral incision, extending laterally from the midline immediately below the forelimb.</li>
		<li>Make a diagonal 1 cm incision between the hindlimb and genitals.</li>
		<li>Make small cuts along the midline skin incisions to peel away the associated connective tissue and expose the subcutaneous adipose depot. Pin the skin down.</li>
		<li>Identify the &#34;C-shaped&#34; subcutaneous adipose tissue and artery/vein that runs perpendicular to the abdominal cavity.</li>
		<li>Start from the bottom of the C shape near the genitals and gently grasp the adipose tissue to expose the connective tissues. Make small cuts in the connective tissue to separate the fat from the skin. Avoid disturbing the exposed vasculature.</li>
		<li>Continue dissecting away the connective tissue until the subcutaneous adipose tissue can be removed intact. Carefully separate the exposed artery from the surrounding connective tissue.</li>
		<li>Store the isolated subcutaneous adipose in HEPES buffer on ice until ready to isolate the vascular bed of interest.</li>
		<li>Repeat steps 1.3.3.-1.3.5. for the remaining posterior subcutaneous adipose depot.</li>
	</ol>
	</li>
	<li>Isolation of the mesenteric adipose depot 
	NOTE: Dissecting tools needed: straight Bonn scissors (9 cm), Graefe forceps, a pair of #5 forceps, straight iris scissors, and curved Bonn scissors (9 cm).
	<ol>
		<li>Use the Graefe forceps to lift the thin peritoneal cavity wall above the urinary bladder and make a small incision using straight scissors.</li>
		<li>Slowly lift the penetrated peritoneal cavity wall, carefully insert the straight iris scissors into the incision site, and make a 4-5 cm incision from the urinary bladder to the sternum.</li>
		<li>Make two 1 cm long horizontal incisions with the straight iris scissors, extending laterally from the midline to immediately above the hindlimb and below the forelimb. Use the Graefe forceps to peel back the abdominal musculature and expose the peritoneal viscera.</li>
		<li>Repeat step 1.4.3. on the opposite side.</li>
		<li>Use the pair of #5 forceps to lift the intestines out of the visceral cavity to reveal the mesenteric adipose.</li>
		<li>Use the curved scissors and #5 forceps to separate the adipose along the colon, starting from the caecum to where the colon descends from view.</li>
		<li>Return to the caecum and use the curved scissors and #5 forceps to separate the adipose along the small intestine to the pancreas. Remove a small part of the pancreas to completely isolate the mesenteric adipose. 
		&#8203;NOTE: Removing a small part of the pancreas along with the mesenteric adipose may help in cleaning the arteries as it provides stability during cleaning.</li>
		<li>Store the isolated mesenteric adipose in HEPES buffer on ice until ready to isolate the mesenteric arteries.</li>
	</ol>
	</li>
	<li>Isolation of the respective arteries from subcutaneous and mesenteric adipose tissues 
	NOTE: Dissecting tools needed: a dissecting dish and stereoscope with a light source, a pair of #55 forceps, and a pair of #5 forceps.
	<ol>
		<li>Place ~10 mL of cold HEPES buffer into the dissecting dish and transfer the adipose tissue to the dish using the #5 forceps.</li>
		<li>Use the stereoscope at 1x magnification to view and position the adipose tissue to expose the artery/vein pair(s) (Figure 2).</li>
		<li>Use the #55 forceps to carefully remove the parenchymal adipose from the artery. Remove small pieces of adipose at a time and carefully observe artery/vein pairs under the stereoscope to identify and avoid damaging the vasculature of interest. 
		NOTE: Adjust the magnification and light source accordingly to clean the arteries. Increase the magnification and light intensity to better view the arteries as adipose is removed. Fine cleaning of the arteries should be done under 4x-5x magnification. It is important to distinguish between connective tissues (collagens), veins, and arteries. Connective tissues and collagen fibers have a string-like appearance without branches, are striated, and have a whitish color. Unlike collagen fibers and connective tissues, arteries and veins are transparent and have multiple branches with a defined lumen. Small arteries and veins may appear similar in appearance when free of parenchymal tissue. The presence of blood in the lumen can further help one to identify the arteries from veins. Veins have thinner blood vessel walls and a larger lumen and contain more blood compared to comparably sized arteries.</li>
		<li>Repeat step 1.5.3. until the arteries are completely void of parenchymal adipose tissue.</li>
		<li>Use #5 forceps to transfer the cleaned arteries to a new tube with fresh HEPES buffer and keep on ice until ready for digestion.</li>
	</ol>
	</li>
</ol>
2. Digestion of isolated arteries for isolation of endothelial cells
NOTE: Please refer to Figure 2 for the schematic of tissue digestion and isolation of endothelial cells and the Table of Materials for a complete list of supplies needed for the protocol.
<ol>
	<li>Add 1 mg each of dispase and elastase to each 2 mL microcentrifuge tube. Add 2 mL of dissociation solution to each tube. Vortex and incubate at 37 &#176;C until completely dissolved. Use #5 forceps to transfer the cleaned arteries (step 1.5.5.) to the respective sample tubes. The final concentration of each enzyme is 0.5 mg/mL.</li>
	<li>Incubate at 37 &#176;C for 1 h with gentle agitation by inversion every 10-15 min, such that the arteries are suspended in solution to maintain consistent and uniform contact between the enzymes and arteries.</li>
	<li>Weigh 1 mg of collagenase type I per sample and add it to new tubes.</li>
	<li>Allow the arteries to settle at the bottom of the tube. Remove 500 &#181;L of the buffer from the top without disturbing the arteries and transfer it into designated tubes containing collagenase. Vortex and return the solution to the respective samples in the original sample tubes. The final concentration of collagenase type I is 0.5 mg/mL.</li>
	<li>Incubate at 37&#160;&#176;C for 15 min. Ensure that the arteries separate into pieces following brief shaking of the sample tube. If the arteries do not separate into pieces following perturbation, incubate in 5 min increments until the arterial breakdown is visible.</li>
	<li>Using a glass pipette, vigorously triturate the digested tissue 10x-15x to mechanically dissociate the cells.</li>
	<li>Pass the solution and digested tissue through a 70 &#181;m cell strainer or a flow cytometry tube strainer to obtain single-cell suspensions.</li>
</ol>
3. Staining of endothelial cells prior to flow cytometry
NOTE: The staining and processing of endothelial cells are carried out on ice to improve cell viability, unless instructed. The 5 mL polystyrene round-bottom tubes are centrifuged at 1,163 x g for 3 min at room temperature (RT).
<ol>
	<li>Centrifuge the single-cell suspensions at 1,163 x g for 3 min at RT.</li>
	<li>Remove the supernatant and resuspend the cell pellet in 1 mL of PBS + 5% BSA for 30 min at RT. 
	NOTE: Researchers should consider blocking FC receptors depending on the cell type, target of interest, and antibody used18,19.</li>
	<li>Add 1 &#181;L of cell viability stain (405 nm excitation) (Table of Materials) and incubate in the dark for 30 min at RT.</li>
	<li>Centrifuge the cell suspension at 1,163 x g for 3 min at RT. Resuspend the cell pellet in 200 &#181;L of PBS + 1% BSA.</li>
	<li>To the samples, add primary antibodies conjugated to CD31 (CD31-PE, 0.75 &#181;g/sample) and CD45 (CD45-FITC, 2.5 &#181;g/sample) and incubate on a rocker in the fridge for 15 min after covering the samples with aluminum foil. 
	NOTE : To identify and/or obtain a pure population of endothelial cells, probe for both CD31 and CD45 using CD31- and CD45-conjugated primary antibodies with distinct excitation/emission fluorescence and gate/sort cells with a CD31+CD45&#8722; expression profile. Compensation may be required depending on the fluorophores used18,20,21.</li>
	<li>Wash the cells by adding 1 mL of PBS + 1% BSA directly to the cell suspension.</li>
	<li>Centrifuge the cell suspension at 1,163 x g for 3 min at RT. Remove the supernatant and resuspend in 200 &#181;L of 1% formaldehyde with 0.1% BSA. Store in the fridge covered with aluminum foil until ready for flow cytometry. 
	NOTE: Cell yields are improved by reducing the wash and centrifugation steps. These may need to be altered depending on the outcome approach. The sample in fixative should be analyzed within 24 h.</li>
</ol>

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The authors have no conflicts of interest.

Endothelial dysfunction is a precursor to severe disease states, likely driving the development of atherosclerosis, hypertension, and stroke3,23. While the identified mechanisms underlying endothelial dysfunction in a given pathological condition are many, distinct vascular beds are likely to be differentially influenced by pathological conditions4,24. Furthermore, different cardiovascular risk factors (e.g., obesity, hypertension, dyslipidemia, smoking, diabetes) induce dysfunction through a variety of distinct mechanisms23,25. Therefore, it is critical to isolate endothelial cell populations from established animal models of disease or accessible human tissue and perform assays on cells immediately removed from the in vivo environment. Isolating cells in this way has a unique advantage over studying cells in culture in that they are void of culture-induced phenotypic changes5,6. Moreover, including a heterogenous endothelial cell population, as observed in vivo26,27 (and that can be further separated using flow-assisted cell sorting), from a live organism better informs the in vivo environment. Finally, this method is applicable to the investigation of numerous animal models and potentially human tissue and can be used to generate primary cell culture lines, if such a need is warranted.
The critical step in this protocol, and the step that will need most adjusting for different vascular beds or cell types not presented here, is the digestion of vascular tissue. This step must be optimized for cell health without diminishing cell yield. The specific enzymes used and the duration for digestion are critical to optimizing cell health and yield to be able to adequately perform downstream assays. For the identification of membrane expression of CD36, as detected by flow cytometry in subcutaneous and mesenteric endothelial cells (Figure 4), a modified version of the digestion protocol originally designed for patch-clamp electrophysiology28 was developed. This included an extension of the collagenase I digestion time to 30 min to increase cell yields to better meet the demands of flow cytometry vs. what is required for patch-clamp studies. As this modification may impact cell health to some extent, a cell viability stain was used in the flow cytometry analyses to ensure that only viable endothelial cells were assessed following this protocol (Figure 3). It is recommended that studies aimed at isolating cells from vascular tissue should include a marker for cell viability prior to assessing the desired approach.
The described protocol is sufficient to liberate viable endothelial cells for analysis and downstream applications from &#8804;1 mg arterial samples derived from mice; however, if the arteries of interest were to be isolated from different tissues or organisms (e.g., humans) that would result in significantly different arterial masses, one would have to optimize the digestion enzyme content and the duration of incubation to efficiently isolate the cell population of interest. For some vascular beds that begin with even smaller amounts of starting tissue than the subcutaneous and mesenteric beds presented here (e.g., coronary arteries), pooling of arteries from several mice may be necessary per sample. Indeed, this may be a necessary step for any given vascular bed given that the outcome approach requires a relatively high cell yield. A major limitation is that, due to the nature of variations per sample digestion, cell yields can sometimes be significantly different across batches even when from the same vascular bed. This can cause issues when analyzing data and should be accounted for via normalization when appropriate. For instance, CD36 expression was extremely variable in the raw data due to alterations in cell yields across individual samples of subcutaneous and mesenteric adipose arteries. Therefore, raw data was normalized to CD31+CD45&#8722; mean fluorescence intensity with the assumption that this method would correct for batch differences (Figure 4). Of course, depending on the approach, more sophisticated statistical analyses and normalization methods may be required.
In summary, this paper presents a method to dissect, isolate, and digest subcutaneous and mesenteric arteries from mice to investigate the expression and/or function of endothelial targets of interest. With modifications to the presented protocol, different vascular beds and cell types (e.g., smooth muscle cells) can be investigated. This protocol, as a foundation for a plethora of available experimental approaches, has the potential to advance the understanding of vascular cell biology and mechanisms of vascular dysfunction.

Endothelial cells are well recognized for their important roles in a variety of physiological processes, including barrier functions, angiogenesis, and vascular tone regulation1,2. Although endothelial cell dysfunction is well documented in promoting atherosclerosis, hypertension, diabetes, etc., the underlying mechanisms driving endothelial dysfunction remain poorly understood and likely differ between distinct vascular beds2,3,4. The effort to unravel these pathological mechanisms of endothelial cell dysfunction is challenged by the limited access to a pure population of endothelial cells from tissues and/or the phenotypic changes of endothelial cells in culture5,6. Therefore, being able to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease.
This protocol presents the procedure to dissect subcutaneous and mesenteric adipose tissues from mice, followed by isolation of their respective arterial vasculature. Functionally viable endothelial cells lining the arterial walls are liberated using a specific cocktail of enzymes. Special care was taken to optimize the conditions of the digestion protocol to obtain a sufficient yield of endothelial cells from &#60;1 mg of starting tissue while keeping biomolecular markers intact for analysis.&#160;Isolated endothelial cells are next identified using flow cytometry. The presence of CD31 (PECAM) is used to primarily identify endothelial cells. Due to the expression of CD31 in other cell types, including several of hematopoietic origin that also express CD45, the purity of the isolated endothelial cells was further enhanced by the exclusion of cells expressing both CD31 and CD455,6,7,8. Moreover, depending on the research question and the downstream applications to be employed, researchers should consider selecting an extensive panel of positive and negative selection markers to optimize the purity of the cell population of interest.
Though the technique of dissecting and isolating adipose depot arteries has been used in the previous publications9,10,11,12,13, a detailed protocol describing the isolation of embedded arteries has yet to be presented. Demonstrating the technique for the isolation and digestion of arteries from different vascular beds from a given species of interest will provide options for researchers to evaluate freshly isolated vascular cells&#160;from arteries of interest and allow them to perform a wide range of functional tests on specific cell types. Tests may include but are not limited to flow cytometry for cell sorting and membrane protein expression5,8, electrophysiology for ion channel activity9, molecular profiling (proteomics/genomic analyses, etc.)14,15, and the generation of primary cell lines for drug screening in vitro16,17.

<table><tbody><tr><td>&lt;strong&gt;Tissue dissection tools&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>5 forceps (2)</td><td>Fine Science Tools</td><td>11252-00</td><td>For isolation of mesenteric adipose depot and arteries transfer</td></tr><tr><td>55 forceps (2)</td><td>Fine Science Tools</td><td>11295-51</td><td>For parenchymal adipose removal</td></tr><tr><td>Curved Bonn scissors</td><td>Fine Science Tools</td><td>14061-10</td><td>For isolation of mesenteric adipose depot</td></tr><tr><td>Graefe forceps</td><td>Fine Science Tools</td><td>11051-10</td><td>For general dissection steps and isolation of subcutaneous adipose depot</td></tr><tr><td>Straight Bonn scissor</td><td>Fine Science Tools</td><td>14060-09</td><td>For general dissection steps and isolation of subcutaneous adipose depot</td></tr><tr><td>Stereoscope with light source</td><td>Laxco</td><td>Z230PT40</td><td>For parenchymal adipose removal and artery isolation</td></tr><tr><td>&lt;strong&gt;Digestive enzymes for Artery Digestion&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Collagenase Type I</td><td>Wothington-BioChem</td><td>LS004194</td><td></td></tr><tr><td>Dispase (Neutral Protease)</td><td>Wothington-BioChem</td><td>LS02110</td><td></td></tr><tr><td>Elastase</td><td>Wothington-BioChem</td><td>LS002292</td><td></td></tr><tr><td>&lt;strong&gt;Solutions Recipes&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;HEPES Buffer, pH 7.4&lt;/em&gt;</td><td></td><td></td><td>&lt;em&gt;Final concentration&lt;/em&gt;</td></tr><tr><td>Calcium chloride dihydrate</td><td>J.T.Baker</td><td>1332-1</td><td>2 mM</td></tr><tr><td>Dextrose (D-glucose) anhydrous</td><td>Fisher</td><td>D16-500</td><td>10 mM</td></tr><tr><td>HEPES</td><td>Fisher</td><td>BP310-500</td><td>10 mM</td></tr><tr><td>Magnesium Chloride</td><td>Fisher</td><td>M33-500</td><td>1 mM</td></tr><tr><td>Potassium Chloride</td><td>Fisher</td><td>P217-500</td><td>5 mM</td></tr><tr><td>Sodium chloride</td><td>Oxoid</td><td>LP0005</td><td>145 mM</td></tr><tr><td>&lt;em&gt;Dissociation Solution, pH 7.30-7.40&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Dextrose (D-glucose) anhydrous</td><td>Fisher</td><td>D16-500</td><td>10 mM</td></tr><tr><td>HEPES</td><td>Fisher</td><td>BP310-500</td><td>10 mM</td></tr><tr><td>Magnesium Chloride</td><td>Fisher</td><td>M33-500</td><td>2 mM</td></tr><tr><td>Potassium Chloride</td><td>Fisher</td><td>P217-500</td><td>56 mM</td></tr><tr><td>Sodium chloride</td><td>Oxoid</td><td>LP0005</td><td>55 mM</td></tr><tr><td>Sodium L-Glutamate monohydrate</td><td>TCI</td><td>G0188</td><td>80 mM</td></tr><tr><td>&lt;strong&gt;Flow Cytometry Analyses&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>5 mL Polystyrene round-bottom tube with cell-strainer cap</td><td>Falcon</td><td>352235</td><td></td></tr><tr><td>CD31-PE</td><td>Miltenyi Biotec</td><td>130-119-653</td><td>0.75&amp;micro;g/sample</td></tr><tr><td>CD36-APC</td><td>R&amp;amp;D Systems</td><td>AF2519</td><td>2.5&amp;micro;g/ sample</td></tr><tr><td>CD45-FITC</td><td>BioLegend</td><td>103108</td><td>2.5&amp;micro;g/ sample</td></tr><tr><td>Live/Dead Fixable Violet Dead Cell Staining kit (cell viability stain)</td><td>Invitrogen</td><td>L34955</td><td>1&amp;micro;L/sample</td></tr></tbody></table>

isolation and identification of vascular endothelial cells from distinct adipose depots for downstream applications

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45&#8722; cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cells&#160;from arteries of interest and allow them to perform a wide range of functional tests on specific cell types.

A schematic of the workflow is shown in Figure 1. The schematic highlights the protocol steps described in more detail in the protocol text. Figure 2 shows pictures of subcutaneous adipose (Figure 2A, left) following dissection and a subcutaneous artery arcade (Figure 2A, right) following cleaning of the parenchymal tissue. Figure 2 also shows the mesenteric adipose (Figure 2B, left) following dissection and the mesenteric arterial arcade (Figure 2B, right) following cleaning of the parenchymal tissue.
The protocol presented here is designed to assist researchers potentially interested in a) comparing distinct adipose depot vasculature beds in basic science approaches and/or disease models, b) the digestion of vascular beds prior to isolation of vascular cells of interest for downstream application, and c) the use of flow cytometry to identify vascular cells of interest (Figure 3) for a variety of applications including, but not limited to, protein expression analyses as performed here (Figure 4). Figure 3 details an example of our approach to identify endothelial cells from digested mouse arteries using flow cytometry. Cell preparations obtained from digested subcutaneous (Figure 3A) or mesenteric (Figure 3B) adipose arteries were stained with CD31-PE, CD45-FITC, and a cell viability dye prior to the fixation and identification of viable CD31+CD45&#8722; endothelial cells using flow cytometry, as similarly performed elsewhere8. The cell viability stain allowed for the identification of only those viable cells that survived the isolation and digestion protocol prior to fixation. The use of this method will allow researchers to assess the expression or function of viable vascular cells of interest.
To determine if endothelial cells isolated from distinct adipose depot vasculature exhibited differences in membrane protein expression, isolated cells were probed for the fatty acid translocase, CD36 (Figure 4), as this membrane protein was recently shown to be essential in the endothelial-mediated distribution of fatty acids to tissues22. Therefore, determining the relative expression differences between membrane CD36, for example, in distinct vascular beds may a) support existing data regarding differential preference for fatty acid utilization among different tissues and/or b) unveil potential novel differences in tissue distribution of fatty acids in health and disease. From the same preparations of digested vascular cells exemplified in Figure 3, CD31+CD45&#8722;CD36+ endothelial cells were identified in subcutaneous (Figure 4A) and mesenteric (Figure 4B) adipose, and CD36 expression was quantified in this population in each vascular bed. Figure 4C and Figure 4D reveal that both the percentage of endothelial cells expressing CD36 and the intensity of CD36 expression were greater in subcutaneous endothelial cells compared to that observed in mesenteric endothelial cells. These preliminary findings support the use of our methodology to identify distinct differences between vascular beds.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63999/63999fig01.jpg" /> 
Figure 1: Working scheme to isolate endothelial cells from subcutaneous and visceral adipose tissues for downstream applications. (A)10-12-week-old C57BL/6J mice were euthanized and used to demonstrate this procedure. (Bi) First, the subcutaneous adipose is removed. (Bii) Then, the mesenteric (visceral) adipose tissue is subsequently removed. The white dashed lines indicate the locations of the subcutaneous (left) and mesenteric (right) adipose depots following the dissection and removal. The respective arteries are isolated for downstream applications. (C) In this protocol, isolated arteries are next digested using a specific enzyme cocktail as a first step in liberating functionally viable endothelial cells. (D) Isolated endothelial cells are identified by probing for CD31+ and CD45&#8722; cells using conjugated antibodies prior to flow cytometry analysis. Cells with an expression profile of CD31+/CD45&#8722; are the endothelial cells of interest for additional analyses. <a href="https://www.jove.com/files/ftp_upload/63999/63999fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63999/63999fig02.jpg" /> 
Figure 2: Isolated subcutaneous and visceral adipose tissues and respective arteries. (A) Left: Isolated &#34;C-shaped&#34; subcutaneous adipose tissue from hindlimbs. A major branch of the subcutaneous adipose artery, denoted by arrow (1), is revealed when parenchymal adipose is removed. Right: Isolated subcutaneous adipose arteries. (B) Left: The mesenteric (visceral) adipose tissue is isolated from the gut. Right: The respective arterial arcade is isolated by removing the parenchymal tissue. Different scales are used between the pictures of adipose (5 mm) and arteries (1 mm). <a href="https://www.jove.com/files/ftp_upload/63999/63999fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63999/63999fig03.jpg" /> 
Figure 3: Identification of endothelial cells following arterial tissue digestion via flow cytometry. Representative plots showing cells liberated from isolated (A) subcutaneous or (B) mesenteric adipose arteries. Cells were exposed to conjugated antibodies that targeted extracellular epitopes of CD31 (PE) and CD45 (FITC) prior to fixation. Flow cytometry was used to identify the CD31+CD45&#8722; endothelial cell population. A cell viability stain was used to select only the cells that survived the isolation and digestion protocols for subsequent analyses. In addition, appropriate gating at this stage will further separate an intended cell population from contaminating cells should the size of the cell type of interest be known. <a href="https://www.jove.com/files/ftp_upload/63999/63999fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63999/63999fig04.jpg" /> 
Figure 4: Analysis of CD36 membrane expression in subcutaneous vs. mesenteric adipose artery endothelial cells via flow cytometry. Representative population plots and histograms showing endothelial CD36 (APC) membrane expression in (A) subcutaneous or (B) mesenteric adipose arteries. (C) Percentage of endothelial cells (ECs) expressing CD36 in subcutaneous vs. mesenteric ECs (n = 5, 3 males, 2 females; *p &#60; 0.05 following Student&#39;s t-test). (D) Normalized EC membrane CD36 expression in subcutaneous vs. mesenteric adipose arteries (n = 5, 3 males, 2 females; *p &#60; 0.05 following Student&#39;s t-test). <a href="https://www.jove.com/files/ftp_upload/63999/63999fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications at JoVE.com

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

This protocol details a method for the dissection of mouse adipose depots and the isolation and digestion of respective arteries to liberate and then identify the endothelial cell population. Freshly isolated cells used in downstream applications will advance the understanding of vascular cell biology and the mechanisms of vascular dysfunction.

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone ...

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Research

JoVE Journal

Biology

2.7K Views.  University of Delaware. Dit protocol stelt de onderzoeker in staat om slagaders van belang te ontleden en vasculaire cellen te isoleren om een breed scala aan functionele tests uit te voeren. Onderzoeker kan verschillende vasculaire bedden en celtypen onderzoeken om de kennis van vasculaire celbiologie en het mechanisme van vasculaire disfunctie uit te breiden. Deze methode is het meest toepasbaar op onderzoekers die onderzoek doen naar elementaire vasculaire celbiologie, evenals vasculaire celdisfunctie in diermode...

Video: Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Immunology and Infection

Localization, Identification, and Excision of Murine Adipose Depots

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.

Due to the drastic and negative connection between obesity and other comorbidities, research on the role adipose plays in disease and overall health is warranted. We present a protocol for the isolation and excision of adipose depots allowing for the study of adipose using in situ and in vitro methods.

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 ...

Medicine

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

Bioengineering

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

Three-Dimensional Culture of Vascularized Thermogenic Adipose Tissue from Microvascular Fragments

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for the design of personalized microtissues for health screening and drug testing. Current strategies are often quite complex and fail to accurately fully depict the multicellular and functional properties of thermogenic adipose tissue. Microvascular fragments, small intact microvessels comprised of arteriole, venules, and capillaries isolated from adipose tissue, serve as a single autologous source of cells that enable vascularization and adipose tissue formation. This article describes methods for optimizing culture conditions to enable the generation of three-dimensional, vascularized, and functional thermogenic adipose tissues from microvascular fragments, including protocols for isolating microvascular fragments from adipose tissue and culture conditions. Additionally, best practices are discussed, as are techniques for characterizing the engineered tissues, and sample results from both rodent and human microvascular fragments are provided. This approach has the potential to be utilized for the understanding and development of treatments for obesity and metabolic disease.

Here, we present a detailed protocol outlining the use of microvascular fragments isolated from rodent or human fat tissue as a straightforward approach to engineer functional, vascularized beige adipose tissue.

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for ...

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...

Human SATMVECs: Isolation and Adipocyte Cross-Talk Studies

Studying Adipose Endothelial Cell/Adipocyte Cross-Talk in Human Subcutaneous Adipose Tissue

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. Significant heterogeneity in endothelial cell (EC) genotype and phenotype depends on their vascular bed and host disease state. The ability to isolate MVECs from tissue-specific vascular beds and individual patient groups offers the opportunity to directly compare MVEC function in different disease states. Here, using subcutaneous adipose tissue (SAT) taken at the time of insertion of cardiac implantable electronic devices (CIED), we describe a method for the isolation of a pure population of functional human subcutaneous adipose tissue MVEC (hSATMVEC) and an experimental model of hSATMVEC-adipocyte cross-talk.
hSATMVEC were isolated following enzymatic digestion of SAT by incubation with anti-CD31 antibody-coated magnetic beads and passage through magnetic columns. hSATMVEC were grown and passaged on gelatin-coated plates. Experiments used cells at passages 2-4. Cells maintained classic features of EC morphology until at least passage 5. Flow cytometric assessment showed 99.5% purity of isolated hSATMVEC, defined as CD31+/CD144+/CD45-. Isolated hSATMVEC from controls had a population doubling time of approximately 57 h, and active proliferation was confirmed using a cell proliferation imaging kit. Isolated hSATMVEC function was assessed using their response to insulin stimulation and angiogenic tube-forming potential. We then established an hSATMVEC-subcutaneous adipocyte co-culture model to study cellular cross-talk and demonstrated a downstream effect of hSATMVEC on adipocyte function.
hSATMVEC can be isolated from SAT taken at the time of CIED insertion and are of sufficient purity to both experimentally phenotype and study hSATMVEC-adipocyte cross-talk.

Author Spotlight: Insights into Cardiometabolic Diseases with Subcutaneous Adipose Tissue Microvasculature Studies 

Here, we describe a protocol for isolating, culturing, and phenotyping microvascular endothelial cells from human subcutaneous adipose tissue (hSATMVECs). Additionally, we describe an experimental model of hSATMVEC-adipocyte cross-talk.

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. ...

Isolation and Characterization of Human Adipocyte-Derived Extracellular Vesicles using Filtration and Ultracentrifugation

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can modulate the cellular status of other cells locally in tissue microenvironments or through liberation into peripheral blood. Adipocyte-derived EVs are elevated in the peripheral blood and show alterations in their cargo (RNA and protein) during metabolic disturbances, including obesity and diabetes. Adipocyte-derived EVs can regulate the cellular status of neighboring vascular cells, such as endothelial cells and adipose tissue resident macrophages to promote adipose tissue inflammation. Investigating alterations in adipocyte-derived EVs in vivo is complex because EVs derived from peripheral blood are highly heterogenous and contain EVs from other sources, namely platelets, endothelial cells, erythrocytes and muscle. Therefore, the culture of human adipocytes provides a model system for the study of adipocyte derived EVs. Here, we provide a detailed protocol for the extraction of total small EVs from cell culture media of human gluteal and abdominal adipocytes using filtration and ultracentrifugation. We further demonstrate the use of Nanoparticle Tracking Analysis (NTA) for quantification of EV size and concentration and show the presence of EV-protein tumor susceptibility gene 101 (TSG101) in the gluteal and abdominal adipocyte derived-EVs. Isolated EVs from this protocol can be used for downstream analysis, including transmission electron microscopy, proteomics, metabolomics, small RNA-sequencing,&#160;microarrays and can be&#160;utilized in functional in vitro/in vivo studies.

We describe the isolation of human adipocyte-derived extracellular vesicles (EVs) from gluteal and abdominal adipose tissue using filtration and ultracentrifugation. We characterize the isolated adipocyte-derived EVs by determining their size and concentration by Nanoparticle Tracking Analysis and by western blotting for the presence of EV-protein tumor susceptibility gene 101 (TSG101).

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can ...

Semi-Automated Isolation of Murine White Adipose Tissue

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

Author Spotlight: Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

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