This work was supported by grants from the National Heart, Lung, and Blood Institute of the National Institutes of Health (NIH) (1R01HL160740 and 5 T32GM132054-04) and the Florida Heart Research Foundation.&#160;We would like to thank Kassandra Gomez for her help synthesizing and imaging the hydrogels.

The in vivo work was approved and overseen by the Institutional Animal Care and Use Committee (IACUC) at Florida International University and conformed to current National Institutes of Health (NIH) guidelines. For this protocol, the procedure does not differ depending on the strain, weight, age, and sex of the mouse. The type of calcification being studied, diets, and treatments may alter the length of the study and the weight of the mouse used and may be dependent on a specific strain and sex of mouse used in the study. For this protocol, both male and female C57BL/6J mice were used, and they were fed a calcification diet. The mice were sacrificed between 20 weeks and 24 weeks of age.
1. Isolation and excision of the aorta
<ol>
	<li>Fluorescent markers
	<ol>
		<li>Forty-eight hours before euthanasia, inject the mice with 20 &#181;M OsteoSense 680, a fluorescent imaging agent, at the recommended dose of 100 &#181;L per 25 g via an intravenous injection.</li>
		<li>Under the fume hood, place a Styrofoam board with four t-pins, a bucket lined with a paper towel, a 50 mL tube stuffed with a paper towel, a 1.5 mL microcentrifuge tube, dissection tools (see the Table of Materials), and ethanol in a spray bottle. Place beakers with PBS on ice.</li>
	</ol>
	</li>
	<li>Blood draw
	<ol>
		<li>Add 2 mL of isoflurane into a 50 mL conical tube stuffed with the paper towel. Place the tube in the bucket, and close it with its lid. 
		CAUTION: Isoflurane should be handled in a well-ventilated space with no recirculation of exhaust air due to the toxicity of short-term exposure.</li>
		<li>Transfer one mouse into the bucket at a time. Once sedated, place the mouse on the paper towel in the dorsal position. Holding the mouse with the palm of the non-dominant hand, place the ears back using the thumb and pointer finger.</li>
		<li>Using the tweezers, excise one of the eyes. Hold the mouse above the 1.5 mL tube to begin blood collection, during which 1 mL of blood should be collected. Once the blood stops draining, place the mouse back in the bucket for further sedation.</li>
	</ol>
	</li>
	<li>Euthanasia
	<ol>
		<li>Place a paper towel on the dissection mat. Once the mouse is sedated, place the mouse on the mat in a supine position. Secure the mouse in the supine position using a t-pin to hold down each limb.</li>
		<li>Spray down the abdomen of the mouse with alcohol. Using the tweezers, lift the skin at the chest region. With the scissors, cut down the medial line into the thoracic cavity. Once the sternum has been cut down the center, cut the diaphragm on either side of the ribcage.</li>
		<li>If necessary, cut away the frontal portion of the ribcage to expose the heart and lungs.</li>
	</ol>
	</li>
	<li>Organ removal
	<ol>
		<li>Using tweezers, lift the skin and make an incision down the midline. Remove any excess skin or fat. Remove the reproductive organs and bladder, as well as any fat outside of the midline.</li>
		<li>Move the gastrointestinal (GI) organs to the right-hand side, and follow the intestines in the midline. Once found, lift the midline, and excise the GI tract up to the stomach.</li>
		<li>Excise the kidneys by lifting them away from the midline. Cut as close to the kidney as possible.</li>
		<li>Next, begin the removal of the liver by lifting the lobes and cutting away from the midline.</li>
		<li>Perfuse the heart and aorta. Using a 10 mL syringe, inject cold PBS into the right ventricle. Wait for the lungs to inflate and turn white. Remove the lungs, as well as any excess diaphragm or ribcage.</li>
	</ol>
	</li>
	<li>Bone removal
	<ol>
		<li>Open a pair of scissors wide, and place them at the lower region of the mouse above the tail. Cut downward, and begin lifting the spine from the skin. Remove the fat from the sides of the midline. Once the skin is detached, excise the spine and intact organs by cutting right above the heart. If transporting to a stereoscope, place the spine and intact organs in a beaker of PBS in the ice bucket.</li>
		<li>Move the spine and organs into the dissecting dish. Fill with ice-cold PBS. Pin down the spine and ribcage using needles.</li>
		<li>Under the dissection microscope, use tweezers to lift the heart carefully, and begin cutting above the spine and underneath the aorta. Continue this until the bottom of the spine is reached. Remove the spine from the dissecting dish, and pin down the heart and any extraneous fat or muscle.</li>
	</ol>
	</li>
	<li>Microdissection
	<ol>
		<li>Starting from the area right below the heart, identify the three tubular structures: the esophagus, vena cava, and aorta. Remove the esophagus and vena cava to have a clear visual field of the aorta.</li>
		<li>Using the microdissection forceps and scissors, begin lifting the adipose tissue and cutting as close to the aorta as possible. Be sure not to cut the aorta. Continue this down the medial line until all the aorta as well as the femoral arteries are exposed.</li>
		<li>At the heart, remove all the adipose tissue, exposing the three branches at the aortic arch. Remove the aortic root from the left ventricle by inserting the microdissection scissors into the left ventricle and cutting the muscle surrounding the aorta.</li>
		<li>Once the aorta is isolated and cleaned, image the aorta using a near-infrared scanner to visualize vascular calcification.</li>
		<li>Use the custom MATLAB script (Supplemental File 1) to quantify the total signal of the calcium tracer, which is normalized to the total scanned aorta area. The MATLAB script functions using the Bioinformatics toolbox add-in. MATLAB is first directed to the location of the TIFF files from the near-infrared scan of the aortas, the individual files are then opened, and the pixel intensity values are extracted from the TIFF files.
		<ol>
			<li>Once the image is presented to the user with a color map, select the aorta with the greatest calcification as the scale maximum.</li>
			<li>When MATLAB asks &#34;How many specimens are in the image?&#34; in the command window, input the number of aortas in the current image, and select each aorta one by one. Once an aorta is selected, MATLAB will create a binary image with a mask of the total area of the aorta and another binary image with a mask of the calcified area of the aorta. The values from these masked images are then used to determine the total calcified area, the total area of the aorta, the percentage area positive for calcification, and the mean intensity of the calcified area.</li>
			<li>To select an aorta, make a box around the aorta of interest on the most recent figure on the screen by selecting four corners with one left click and creating a box around the image. To close the box, double-click on the first corner. 
			&#8203;NOTE: The threshold may be different depending on the model of calcification used. It is up to the user to determine what value determines a positive signal for vascular calcification. Once the threshold is set for an experiment, it must remain the same throughout the duration of the experiment.</li>
		</ol>
		</li>
		<li>Once the quantitative data are extracted, perform a one-way ANOVA with Tukey&#39;s post-hoc test to show significance between the groups.</li>
	</ol>
	</li>
</ol>
2. Isolating and extracting EVs from aortas
NOTE: Once the aortas have been isolated and removed, EVs can be extracted from the tissue. Using a digestive solution and multiple centrifuge cycles, EVs can be collected and used for many different techniques, including calcification assays, gel electrophoresis, and immunoblotting13. The protocol for isolating and extracting EVs is as follows:
<ol>
	<li>Immediately after scanning the aortas, pool two to three aortas to yield a sufficient protein concentration.</li>
	<li>Prepare a digestive solution containing 0.25 M sucrose, 0.12 M sodium chloride (NaCl), 0.01 M potassium chloride (KCl), 0.02 M Tris hydrochloride, and 600 U/mL collagenase13.</li>
	<li>Incubate two to three pooled aortas in 1.5 mL of digestive solution for 2 h at 37 &#176;C. Collect the solution. Centrifuge the solution at 1,000 &#215; g for 15 min to remove cell debris.</li>
	<li>Centrifuge the supernatant for an additional 30 min at 33,000 &#215; g to remove microvesicles. Collect and assess the supernatant for calcification potential (see section 3). 
	NOTE: Follow the subsequent steps if gel electrophoresis and protein immunoblotting of the EVs are to be performed.</li>
	<li>Ultracentrifuge the supernatant at 100,000 &#215; g for 1 h to isolate the EVs of interest.</li>
	<li>While the supernatant is undergoing ultracentrifugation, prepare RIPA lysis and extraction buffer (see the Table of Materials) by adding the protease inhibitor and vortexing until dissolved.</li>
	<li>Once the ultracentrifugation is complete, aspirate the supernatant, leaving the pellet, which contains the EVs. Suspend the pellet in the prepared RIPA lysis and protease inhibitor mixture. The samples are ready for gel electrophoresis and western blotting.</li>
</ol>
3. Assessing the calcification potential of extracellular vesicles with light scattering absorbance
NOTE: To measure the real-time mineral formation of EVs, we used an assay originally developed to study mineral formation from growth plate cartilage EVs from cell culture14. Since calcium phosphate deposition is the hallmark of calcification, an increase in light scattering absorbance as the result of calcium phosphate compound formation is indicative of the calcification potential14. In vitro VSMC models are convenient for measuring the calcification potential of EVs. In this technique, a plate reader with a short wavelength filter can quantify the in vitro calcification of EVs. The absorbency reading is recorded at 340 nm, and a higher absorbance is indicative of more calcium phosphate mineral formation. The protocol for the light scattering absorbance assay is as follows:
<ol>
	<li>Centrifuge the conditioned collected medium of VSMCs at 1,000 &#215; g for 5 min to remove cell debris14.</li>
	<li>Centrifuge the supernatant at 33,000 &#215; g for 30 min, collect the supernatant, and transfer it into a new tube. 
	NOTE: The presence of EVs in the collected supernatant can be confirmed through dynamic light scattering (DLS) or nanoparticle tracking analysis (NTA)</li>
	<li>Prepare a 300 mM sodium phosphate monobasic (NaH2PO4) sterile solution. Filter the solution to ensure sterile conditions if necessary.</li>
	<li>Add 1% (v/v) of 300 mM NaH2PO4 to the EV samples, and gently pipette the solution to mix well. Transfer 200 &#181;L of the mixture solution to a 96-well plate. Incubate the 96-well plate in the microplate reader at 37 &#176;C.</li>
	<li>Set the plate reader to record the absorbance at a wavelength of 340 nm every 1 min.</li>
</ol>
4. Assessing the calcification mineral formation of extracellular vesicles with collagen hydrogels
NOTE: The aggregation of EVs and the formation of microcalcifications are observed through collagen hydrogels. These hydrogels act as a scaffold that mimics the collagen density observed in vivo15. This demonstrates the effect of collagen on calcification growth. The protocol for assessing the mineral formation of EVs in hydrogels is as follows:
<ol>
	<li>Day 1
	<ol>
		<li>Prepare a 2.5 mg/mL collagen solution in DMEM. Slowly add 5 M sodium hydroxide to the collagen/DMEM mixture until the color changes to red. Check that the pH of the solution is approximately 7. 
		NOTE: If the solution turns purple and becomes too basic, restart the process.</li>
		<li>Pipette 300 &#181;L of the 2.5 mg/mL collagen solution with a pH of 7 into each well of an 8-well chamber glass. Incubate at 37 &#176;C and 5% CO2 controlled humidity for 72 h.</li>
	</ol>
	</li>
	<li>Day 4
	<ol>
		<li>Add 300 &#181;L of DMEM as a control into the wells.</li>
		<li>Add 300 &#181;L of the EV medium samples collected previously to the remaining wells.</li>
		<li>Incubate at 37 &#176;C and 5% CO2 controlled humidity for 6 days.</li>
	</ol>
	</li>
	<li>Day 10
	<ol>
		<li>Prepare 2.5 &#181;L of Osteosense 680EX with 22.5 &#181;L of DMEM.</li>
		<li>Pipette 2.5 &#181;L of the Osteosense and DMEM mixture into each well. Incubate at 37 &#176;C and 5% CO2 controlled humidity for 24 h.</li>
	</ol>
	</li>
	<li>Day 11
	<ol>
		<li>Image the hydrogels with filters appropriate for Osteosense fluorescence. Image them through the bottom of a coverglass chamber using an inverted microscope or from the top with a long working distance objective.</li>
		<li>Assess the number of calcifications and the calcification size in the collected images using the Particle Analysis options available in ImageJ.
		<ol>
			<li>Use the Image | Adjust | Threshold command to binarize the images such that only the Osteosense signal appears white. Use the Analyze | Analyze particles command to obtain information for each calcification in the image. Be sure to use the same threshold parameters for each image analyzed for consistency.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Bakhshian Nik, A., Hutcheson, J. D., Aikawa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+as+mediators+of+cardiovascular+calcification.">Extracellular vesicles as mediators of cardiovascular calcification.</a> Frontiers in Cardiovascular Medicine. 4, 78 (2017).</li><li>Ho, C. Y., Shanahan, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medial+arterial+calcification:+an+overlooked+player+in+peripheral+arterial+disease.">Medial arterial calcification: an overlooked player in peripheral arterial disease.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 36 (8), 1475-1482 (2016).</li><li>Marinelli, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+arterial+media+calcification+in+chronic+kidney+disease.">Diagnosis of arterial media calcification in chronic kidney disease.</a> Cardiorenal Medicine. 3 (2), 89-95 (2013).</li><li>Moe, S. M., Chen, N. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+vascular+calcification+in+chronic+kidney+disease.">Mechanisms of vascular calcification in chronic kidney disease.</a> Journal of the American Society of Nephrology. 19 (2), 213-216 (2008).</li><li>Mir, B., Goettsch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+as+delivery+vehicles+of+specific+cellular+cargo.">Extracellular vesicles as delivery vehicles of specific cellular cargo.</a> Cells. 9 (7), 1601 (2020).</li><li>Ruiz, J. L., Hutcheson, J. D., Aikawa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+calcification:+Current+controversies+and+novel+concepts.">Cardiovascular calcification: Current controversies and novel concepts.</a> Cardiovascular Pathology. 24 (4), 207-212 (2015).</li><li>New, S. E., Aikawa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+extracellular+vesicles+in+de+novo+mineralization:+An+additional+novel+mechanism+of+cardiovascular+calcification.">Role of extracellular vesicles in de novo mineralization: An additional novel mechanism of cardiovascular calcification.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 33 (8), 1753-1758 (2013).</li><li>Aikawa, E., Hutcheson, J. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphate+feeding+induces+arterial+medial+calcification+in+uremic+mice:+role+of+serum+phosphorus,+fibroblast+growth+factor-23,+and+osteopontin.">Phosphate feeding induces arterial medial calcification in uremic mice: role of serum phosphorus, fibroblast growth factor-23, and osteopontin.</a> Kidney International. 75 (12), 1297-1307 (2009).</li><li>Veseli, B. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+atherosclerosis.">Animal models of atherosclerosis.</a> European Journal of Pharmacology. 816, 3-13 (2017).</li><li>Chen, N. 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The authors have no conflicts of interest to disclose.

When performing the protocol, it is important to note the critical steps for obtaining successful results. During the isolation of the murine aorta, it is vital that the perfusion is performed properly. When injecting the PBS, care must be taken not to puncture the right ventricle. This would cause the liquid to leak directly out of the ventricle and fail to circulate through the lungs, leaving blood within the aorta. Once the perfusion has been conducted properly and microdissection has begun, all the adipose and fatty ...

Calcification is the most significant predictor of cardiovascular disease mortality and morbidity1. Calcification alters the arterial wall mechanics due to the buildup of calcium and phosphate minerals2. In atherosclerosis, calcification can exacerbate local stress and result in plaque rupture, which is the leading cause of heart attacks. Medial calcification-often resulting from chronic kidney disease-is more widespread and leads to significant arterial stiffening, dysfunction, and cardiac overload2,3. Currently, there are no therapeutic options for the treatment or prevention of vascular calcification.
Vascular smooth muscle cells (VSMCs) adopt an osteoblast-like phenotype and release calcifying extracellular vesicles (EVs) that nucleate nascent minerals, thus driving calcification4,5,6. This process resembles the physiological mineralization of osteoblasts in bone7. Although the endpoint of mineralization is similar in the vascular wall and bone matrix, the mechanisms by which the calcifying EVs originate differ in the two tissues8. There are many types of models that are used to study vascular calcification. In vitro, cell culture models mimic the osteogenic transition of VSMCs and subsequent mineral formation with specialized media.
When studying in vivo calcification, the model used depends upon the type of calcification being studied. Hyperlipidemic mouse models are often used to study atherosclerotic calcification, which appears more focal within lipid-rich plaques9. In contrast, medial calcification is more widespread throughout the vasculature and is often studied using chronic kidney disease models that employ an adenine-rich diet regimen to induce renal failure or surgical techniques to remove significant portions of the kidneys10,11. Aggressive models of vascular calcification have used a combination of both hyperlipidemic and chronic kidney disease models12. This protocol provides a method for assessing vascular calcification in murine aortas for both medial and atherosclerotic calcification, extracting EVs from the aortic wall, and observing the mineralization potential in the EVs obtained from in vitro cell culture models. Future studies can use these procedures in mechanistic analyses of vascular calcification and to assess potential therapeutic interventions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>8-well chambered coverglass</td>
			<td>Thermo Scientific</td>
			<td>155409PK</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Syringe</td>
			<td>BD</td>
			<td>302995</td>
			<td></td>
		</tr>
		<tr>
			<td>20 G 1 inch Needle</td>
			<td>BD</td>
			<td>305175</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen, High Concentraion, Rat Tail</td>
			<td>Corning</td>
			<td>354249</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Worthington Biochemical</td>
			<td>LS004174</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Forceps</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-8254</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Dish</td>
			<td>Living Systems Instrumentation</td>
			<td>DD-90-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Pan and Wax</td>
			<td>United Scientific Supplies</td>
			<td>DSPA01-W</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Cytiva</td>
			<td>SH30022.FS</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Sigma-Aldrich</td>
			<td>26675-46-7</td>
			<td></td>
		</tr>
		<tr>
			<td>LI-COR Odyssey</td>
			<td>LI-COR</td>
			<td>DLx</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting Curved Scissors (24 mm Blade)&#160;</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5913</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting Spring Scissors (13 mm Blade)</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5677</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting Spring Scissors (5 mm Blade)</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5600</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Dissecting Tweezers (0.10 x 0.06 mm Tip)</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-4976</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima MAX-TL Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>B11229</td>
			<td></td>
		</tr>
		<tr>
			<td>OsteoSense 680EX</td>
			<td>Perkin Elmer</td>
			<td>NEV10020EX</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce Protease Inhibitor</td>
			<td>Thermo Scientific</td>
			<td>A32963</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fischer Chemical</td>
			<td>P217</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA Lysis and Extraction Buffer</td>
			<td>G Biosciences</td>
			<td>786-489</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fischer Chemical</td>
			<td>BP358</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Thermo Scientific</td>
			<td>A4782602</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>S0751</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S7903</td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy HTX Multimode Reader</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture plate, 96-well</td>
			<td>Thermo Fisher</td>
			<td>167008</td>
			<td></td>
		</tr>
		<tr>
			<td>T-Pins</td>
			<td>United Scientific Supplies</td>
			<td>TPIN02-PK/100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Hydrochloride</td>
			<td>Fischer Chemical</td>
			<td>BP153</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of extracellular vesicle-mediated vascular calcification using in vitro and in vivo models

Cardiovascular disease is the leading cause of death in the world, and vascular calcification is the most significant predictor of cardiovascular events; however, there are currently no treatment or therapeutic options for vascular calcification. Calcification begins within specialized extracellular vesicles (EVs), which serve as nucleating foci by aggregating calcium and phosphate ions. This protocol describes methods for obtaining and assessing calcification in murine aortas and analyzing the associated extracted EVs. First, gross dissection of the mouse is performed to collect any relevant organs, such as the kidneys, liver, and lungs. Then, the murine aorta is isolated and excised from the aortic root to the femoral artery. Two to three aortas are then pooled and incubated in a digestive solution before undergoing ultracentrifugation to isolate the EVs of interest. Next, the mineralization potential of the EVs is determined through incubation in a high-phosphate solution and measuring the light absorbance at a wavelength of 340 nm. Finally, collagen hydrogels are used to observe the calcified mineral formation and maturation produced by the EVs in vitro.

Once the aortas are extracted, imaging using a near-infrared optical scanner shows a visual representation of the aorta as well as the vascular calcification (Figure 1). The pixel intensity values within the scanned fluorescent image represent the distribution of calcification and are shown here using a colored heatmap. Quantification methods include identifying a positive threshold and reporting the percentage area of the aorta with values greater than this threshold value and/or reporting ...

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Analysis of Extracellular Vesicle-Mediated Vascular Calcification Using In Vitro and In Vivo Models

This paper presents the methodology for obtaining and assessing vascular calcification by isolating murine aortas followed by extracting calcified extracellular vesicles to observe the mineralization potential.

Analysis of Extracellular Vesicle-Mediated Vascular Calcification Using In Vitro and In Vivo Models

Cardiovascular disease is the leading cause of death in the world, and vascular calcification is the most significant predictor of cardiovascular events; ...

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Biology

1.5K Views.  Florida International University. This paper presents the methodology for obtaining and assessing vascular calcification by isolating murine aortas followed by extracting calcified extracellular vesicles to observe the mineralization potential.

Video: Analysis of Extracellular Vesicle-Mediated Vascular Calcification Using In Vitro and In Vivo Models

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

In vivo and in vitro Studies of Adaptor-clathrin Interaction

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs are distinguished by a polyhedral lattice of clathrin that coats the vesicle membrane and serves as a mechanical scaffold. Clathrin coats are assembled during vesicle formation from individual clathrin triskelia , the soluble form of clathrin composed of three heavy and three light chain subunits7,8. Because the triskelion does not have the ability to bind to the membrane directly, clathrin-binding adaptors are critical to link the forming clathrin lattice to the membrane through association with lipids and/or membrane proteins9. Adaptors also package transmembrane protein cargo, such as receptors, and can interact with each other and with other components of the CCV formation machinery9.
Over twenty clathrin adaptors have been described, several are involved in clathrin mediated endocytosis and others localize to the trans Golgi network or endosomes9. With the exception of HIP1R (yeast Sla2p), all known clathrin adaptors bind to the N-terminal -propeller domain of the clathrin heavy chain9. Clathrin adaptors are modular proteins consisting of folded domains connected by unstructured flexible linkers. Within these linker regions, short binding motifs mediate interactions with the clathrin N-terminal domain or other components of the vesicle formation machinery9. Two distinct clathrin-binding motifs have been defined: the clathrin-box and the W-box9. The consensus clathrin-box sequence was originally defined as L[L/I][D/E/N][L/F][D/E]10 but variants have been subsequently discovered11. The W-box conforms to the sequence PWxxW (where x is any residue).
Sla1p (Synthetic Lethal with Actin binding protein-1) was originally identified as an actin associated protein and is necessary for normal actin cytoskeleton structure and dynamics at endocytic sites in yeast cells12. Sla1p also binds the NPFxD endocytic sorting signal and is critical for endocytosis of cargo bearing the NPFxD signal13,14. More recently, Sla1p was demonstrated to bind clathrin through a motif similar to the clathrin box, LLDLQ, termed a variant clathrin-box (vCB), and to function as an endocytic clathrin adaptor15. In addition, Sla1p has become a widely used marker for the endocytic coat in live cell fluorescence microscopy studies16. Here we use Sla1p as a model to describe approaches for adaptor-clathrin interaction studies. We focus on live cell fluorescence microscopy, GST-pull down, and co-immunoprecipitation methods.

In vivo and in vitro Studies of Adaptor-clathrin Interaction

Clathrin-mediated endocytosis depends on adaptor proteins that coordinate cargo selection and clathrin coat assembly. Here we describe procedures to study adaptor-clathrin physical interaction and live cell imaging approaches using as a model the yeast endocytic adaptor protein Sla1p.

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs ...

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many degenerative human diseases. The recent discovery of MG53 as a key component of the membrane resealing machinery allows for a better molecular understanding of the basic biology of tissue repair, as well as for potential translational applications in regenerative medicine. Here we detail the experimental protocols for exploring the in vivo function of MG53 in repair of muscle injury using treadmill exercise protocols on mouse models, for testing the ex vivo membrane repair capacity by measuring dye entry into isolated muscle fibers, and for monitoring the dynamic process of MG53-mediated vesicle trafficking and cell membrane repair in cultured cells using live cell confocal microscopy.

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Described here are protocols used to visualize the dynamic process of MG53-mediated cell membrane repair in whole animals and at the cellular level. These methods can be applied to investigate the cell biology of plasma membrane resealing and regenerative medicine.

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Mouse Sperm Cryopreservation and Recovery using the I&amp;middot;Cryo Kit

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields of regenerative medicine, basic cell biology, treatment of diseases, and reprogramming of cells. Here, we describe a step by step protocol for generation of modified mRNA with reduced immune activation potential and increased stability, quality control of produced mRNA, transfection of cells with mRNA and verification of the induced protein expression by flow cytometry. Up to 3 days after a single transfection with eGFP mRNA, the transfected HEK293 cells produce eGFP. In this video article, the synthesis of eGFP mRNA is described as an example. However, the procedure can be applied for production of other desired mRNA. Using the synthetic modified mRNA, cells can be induced to transiently express the desired proteins, which they normally would not express.

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In this video article, we describe the in vitro synthesis of modified mRNA for induction of protein expression in cells.

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a 'sample preparation protocol' for maize materials (i.e., stem, leaf, and root) suitable for ordinary microcomputed tomography (micro-CT) scanning. Based on high-resolution CT images of maize stem, leaf, and root, we describe two protocols for the phenotypic analysis of vascular bundles: (1) based on the CT image of maize stem and leaf, we developed a specific image analysis pipeline to automatically extract 31 and 33 phenotypic traits of vascular bundles; (2) based on the CT image series of maize root, we set up an image processing scheme for the three-dimensional (3-D) segmentation of metaxylem vessels, and extracted two-dimensional (2-D) and 3-D phenotypic traits, such as volume, surface area of metaxylem vessels, etc. Compared with traditional manual measurement of vascular bundles of maize materials, the proposed protocols significantly improve the efficiency and accuracy of micron-scale phenotypic quantification.

We provide a novel method to improve the X-ray absorption contrast of maize tissue suitable for ordinary microcomputed tomography scanning. Based on CT images, we introduce a set of image-processing workflows for different maize materials to effectively extract microscopic phenotypes of vascular bundles of maize.

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a ...

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...