The authors thank Professor Cijiang He and Dr. Fu Jia for their insights in mice endothelial cell isolation and thank Professor Mone Zaidi for providing the PhAMexcised mice and valuable discussions. The authors would also like to acknowledge the Microscopy CORE at the Icahn School of Medicine at Mount Sinai and staff for the guidance we received. This work was supported by grants from National Institutes of Health grant R01DK097253 and Department of Defense CDMRP grant E01 W81XWH2010836 to I.S.D.

All animal procedures described here were approved by the IACUC at Icahn School of Medicine at Mount Sinai. We used three male mice (H-2Kb-tsA58 transgenic mice with photo-activatable mitochondria [PhAM]) purchased from Jackson lab and kept on a normal chow diet.
1. Working conditions and preparations
<ol>
	<li>Clean the working space inside the laminar flow hood using 70% ethanol and UV-C light.</li>
	<li>Prepare the bead&#39;s washing buffers. Make buffer-1 using 0.1 M sodium phosphate at pH 7.4-8.0, buffer-2 using 1x phosphate-buffered saline (PBS) without calcium and magnesium supplemented with 0.1% bovine serum antigen (BSA), 3 mM EDTA, pH 7.4, and buffer-3 using 0.2 M Tris with 0.1% BSA, pH 8.5.</li>
</ol>
2. Magnetic particle concentrator
<ol>
	<li>Preparation of magnetic beads
	<ol>
		<li>In a 1.5 mL microcentrifuge tube, gently mix 200 &#181;L of magnetic beads (4 x 108 beads) with 200 &#181;L of buffer-1. 
		NOTE: Prepare 200 &#181;L of magnetic beads per mouse 1 day before tissue harvesting. The beads will be used to harvest the glomeruli.</li>
		<li>Place the tube in a magnetic particle concentrator for 1 min, then carefully aspirate the supernatant. Remove the tube from the magnet and resuspend the washed beads in 200 &#181;L of buffer-1.</li>
		<li>Place the tube containing the magnetic beads in the magnetic particle concentrator for 1 min and gently aspirate the supernatant.</li>
		<li>Inactivate the free tosyl groups by washing the magnetic beads 2x, 5 min each, using 200 &#181;L of buffer-2 by placing the tube in the magnetic particle concentrator and discarding the buffer.</li>
		<li>Incubate the magnetic beads in 200 &#181;L of buffer-3 for 24 h at 4 &#176;C, or for 4 h at 37 &#176;C, to inactivate the remaining free tosyl groups. Place the tube in the magnetic concentrator for 1 min, gently aspirate the supernatant, and discard it.</li>
		<li>Wash the magnetic beads 1x with 200 &#181;L of buffer-2 for 5 min at 4 &#176;C. Place the tube back in the magnetic concentrator for 1 min and gently aspirate the supernatant. Remove the tube from the magnetic concentrator and resuspend the magnetic beads in 200 &#181;L of buffer-2 to obtain 4 x 108 beads/mL. 
		&#8203;NOTE: At this stage, it is possible to leave the beads at 4&#160;&#176;C for up to 15 days.</li>
	</ol>
	</li>
</ol>
3. Isolation of mouse kidney glomerular cells
<ol>
	<li>Prepare the perfusion material: 30 mL syringe, 25 G butterfly needle. Prepare the isolation material: sterile 10 cm dish, scalpel, magnetic particle concentrator, 40 &#181;m cell strainer, 100 &#181;m cell strainer.</li>
	<li>Prepare 100 mL of 1x Hank&#39;s Balanced salt solution (HBSS) supplemented with 1% antibiotics and adjust the pH to 7.35. Filter the solution with a 0.2 &#181;m filter and keep it under the hood to avoid contamination.</li>
	<li>Mix 200 &#181;L of magnetic beads from step 2.1.6 with 20 mL of 1x HBSS. 
	NOTE: For each mouse, 200 &#181;L of beads diluted in 20 mL of HBSS 1x will be needed.</li>
	<li>Prepare a 1 mL aliquot of collagenase type-II (125 CDU/mg) at 1 mg/mL in 1x HBSS solution, pH 7.35. Pre-warm the RPMI with 10% FCS medium in a water bath at 37 &#176;C. Pre-warm the endothelial cell culture medium at 37 &#176;C (see Table of Materials for details). 
	NOTE: The RPMI will be used to neutralize the collagenase.</li>
	<li>Pre-coat two wells of a 6-well plate with 2 mL of 10 &#181;g/mL collagen IV (1 mL/well) for 45 min at room temperature. Wash the plate 3x with 1x PBS to remove the acetic acid used to dilute the collagen. Use the coated plates immediately or store them at 2-8 &#176;C for up to 4 weeks. 
	NOTE: Sealing the plate(s) with a transparent film is preferred to prevent drying out. The collagen coating facilitates the attachment of cells to the plate.</li>
	<li>Clean and sterilize all the surgical instruments in Figure 2 by using 70% ethanol and subsequently autoclaving them for 30 min. Clean the surgical workspace with 70% ethanol.</li>
	<li>Anesthetize the mice with an intraperitoneal injection of ketamine/xylazine (10 mg/mL ketamine and 1 mg/mL xylazine). To confirm that the animal is fully anesthetized, check for the toe pinch reflex.</li>
	<li>Fix the paws of the mouse with 19 G needles to hold it in place on a dissection platform. Clean the chest and the abdomen with 70% ethanol, and open the abdomen along the chest with scissors&#160;a (Figure 2).</li>
	<li>Insert a butterfly needle into the left ventricle (Figure 3A) and inject about 100 &#956;L of the bead solution (from step 1.2) to expand the blood circulation.</li>
	<li>Carefully break the inferior vena cava below the kidneys with a 19 G needle (Figure 3A). Continue the perfusion initiated in step 3.9. 
	NOTE: Alternatively, a mini pump can be used at speed: 18.0+, time: 5 min. Good perfusion is important to harvest a great number of glomeruli bearing magnetic beads. This is a non-survival/ terminal procedure.&#160;</li>
	<li>Gently remove the fat tissue with tweezers and excise both kidneys with thin surgical scissors c (Figure 2) and put them in a plate with 1 mL of 1x HBSS on ice. Mince the kidney with scissors b (Figure 2) then with a sterile scalpel to 1 mm small pieces as shown in Figure 3B.</li>
	<li>Next, incubate the minced kidney with 1 mL of collagenase type II from step 3.4 for 35 min at 37 &#176;C with gentle tilting using a horizontal shaker. The tissue should be completely digested after this step, as shown in Figure 3C.</li>
	<li>Add 4 mL of RPMI with 10% FBS (from step 3.4) to neutralize the collagenase. Filter the digested tissue through a sterile 100 &#181;m cell strainer into a 50 mL tube. Use the bottom of a sterile 1.5 mL microcentrifuge tube to stir the digested tissue against the strainer to allow the glomeruli to go through.</li>
	<li>Rinse the cell strainer with 15 mL of 1x HBSS and then centrifuge the flow-through at 200 x g for 5 min at room temperature. At this point, the pellet contains three layers; the beads with glomeruli are visible in the middle layer of the tube, the upper layer consists of smaller structures such as tubules, and the bottom layer is debris (Figure 3D).</li>
	<li>Carefully aspirate the supernatant and the top white layer. The remaining pellet contains the beads bearing the glomeruli and debris (Figure 3D). Resuspend the pellet with 1.5 mL of 1x HBSS and transfer into a 2 mL tube.</li>
	<li>Place the tube in the magnetic concentrator (Figure 3E), aspirate the supernatant, and then wash the beads 2x with 1 mL of 1x HBSS; at this stage, put 20 &#181;L of the cell suspension on a slide to verify the presence of glomeruli under the microscope (Figure 3F; a glomerulus [black arrow], and some beads [yellow arrows] in the suspension).</li>
	<li>Carefully aspirate the supernatant, resuspend the pellet in endothelial cell growth medium (Table of Materials), and transfer it to a clean 2 mL tube. Add 2 &#181;L of IFN&#947; (100 U/mL) to the tube and plate the cells on to one well of a 6-well plate. Incubate the cells at 33 &#176;C.</li>
	<li>After 3 days of culture, carefully change the medium by pipetting out 1 mL of the medium and replacing it with 1 mL of fresh media. At this stage, the cells are heterogeneous with a mixture of glomerular cells (Figure 4G). 
	NOTE: In the outgrowing culture, there will be a mix of glomerular cells and some magnetic beads (Figure 4G). Notice that all the cells should show green, fluorescent mitochondria.</li>
	<li>After 7 days of culture, completely aspirate the medium and replace it with a fresh 2 mL of endothelial cell growth medium supplemented with IFN&#947; (100 U/mL). At this stage, the cells start proliferating but are still heterogeneous (Figure 4A).</li>
	<li>After 10 days of culture when the cells reach ~80% confluency, passage the cells using trypsin as described in step 5.1 and then transfer them into a T25 flask coated with collagen IV.</li>
	<li>After 1 week (total ~21 days of culture), passage the cells to a T75 flask coated with collagen IV.</li>
</ol>
4. Preparation of beads for endothelial cell isolation
<ol>
	<li>Start by coating the sheep anti-rat beads with a rat anti-mouse CD31 antibody3,18,19. Use 10 &#181;L of beads for each T75 flask.</li>
	<li>Wash 10 &#181;L of beads 3x with 200 &#181;L of 1x PBS supplemented with 0.1% BSA, 2 mM EDTA, 1% penicillin/streptomycin. Place the tube in the concentrator magnet to pipette the washing buffer each time.</li>
	<li>Resuspend the beads in 200 &#181;L of PBS, 1% BSA, 2 mM EDTA, 1% penicillin/streptomycin. Add 4.8 &#181;L of rat anti-mouse CD31 antibody7,18,19, place the tube on a horizontal shaker and gently mix for 1 h at room temperature, and then keep the tube at 4 &#176;C overnight.</li>
</ol>
5. Isolating the glomerular endothelial cells with CD31-coated beads
<ol>
	<li>Add 3 mL of 0.25% trypsin to a T75 flask and incubate the cells at 37 &#176;C for 5 min. Neutralize the trypsin with 3 mL of endothelial growth medium, transfer the cells to a 15 mL tube, and then centrifuge at 200 x g for 5 min.</li>
	<li>Aspirate the supernatant and wash the cells in 1 mL of PBS containing 1% BSA, 2 mM EDTA, 1% penicillin/streptomycin. Centrifuge at 200 x g for 5 min.</li>
	<li>Resuspend the pellet in 200 &#181;L of coated beads prepared in step 4, and add 800 &#181;L of PBS containing 1% BSA, 2 mM EDTA, 1% penicillin/streptomycin. Incubate the cells for 45 min with continuous shaking at 37 &#176;C, 5% CO2.</li>
	<li>Place the tube in the magnetic concentrator and wash the cells 4x with 1x PBS containing 1% BSA, 2 mM EDTA, 1% penicillin/streptomycin. Resuspend the cells in 3 mL of endothelial growth medium supplemented with 100 U/mL IFN&#947;.</li>
	<li>Plate 1.5 mL of cells/well in collagen IV-coated wells on a 6-well plate. Culture the cells under permissive conditions at 33 &#176;C. 
	NOTE: The isolated cells express simian virus 40 (SV40) large T antigen tsA58, enabling the activation of continuous proliferation at 33 &#176;C (permissive condition).</li>
	<li>After 4 days in culture, remove 750 &#181;L of the medium and replace it with the same volume of fresh medium supplemented with 100 U/mL IFN&#947;.</li>
	<li>After 10 days to 14 days of culture, check for growing CD31 positive GECs colonies (Figure 4C) that express fluorescent mitochondria using an epifluorescence microscope with a 488 nm filter.</li>
	<li>After 21 days of culture, the cells may have reached 80%-90% confluency; transfer them into a T25 flask. After reaching confluency, cells can be maintained with RPMI growth medium and 10% FCS. 
	NOTE: Before reaching confluence, the cells may look heterogeneous (Figure 4B). However, once they are confluent, they acquire a cobble-stone morphology.</li>
	<li>At this stage, the cells could be cryopreserved. Upon reaching 80% confluency, add 3 mL of 0.25% trypsin to the cells and incubate them at 37 &#176;C for 5 min. Neutralize the trypsin with an equal volume of growth medium supplemented with 10% FBS.</li>
	<li>Centrifuge at 200 x g for 5 min. Discard the supernatant and resuspend the cells in 3 mL of freezing medium (RPMI, 20% FBS, 10% DMSO). Aliquot the cells in cryo-tubes and store them in liquid nitrogen vapor temperature.</li>
</ol>
6. Cultivating the mitoDendra2-GECs
<ol>
	<li>Thaw the cells from the cryoaliquot, from step 5.10, in a water bath at 37 &#176;C and transfer them into a 15 mL tube with 10 mL of pre-warmed endothelial growth medium.</li>
	<li>Centrifuge the suspension at 200 x g for 5 min at room temperature. Remove the medium, and transfer the pellet to the collagen IV-coated T25 cm2 cell culture flask with pre-warmed endothelial cell growth medium supplemented with 100 U/mL IFN&#947; and incubate at 33 &#176;C.</li>
	<li>Change the medium the next day. When the cells reach 70%-80% confluency, usually after 5 days, transfer them to a T75 cm2 cell culture flask.</li>
	<li>To differentiate the cells to endothelial phenotype, transfer the cells to non-permissive conditions at 37 &#176;C in growth media without IFN&#947;.</li>
</ol>
7. Passaging of mitoDendra2-GECs
<ol>
	<li>Remove the medium, wash the cells with PBS, and add 3 mL of warm 0.25% trypsin-EDTA solution (T75 cm2 flask), incubate at 37 &#176;C for 5 min.</li>
	<li>When the cells are detached, add 5 mL of growth medium and pipette up and down. Centrifuge the suspension at 200 x g for 5 min at room temperature and transfer the cells to a new collagen IV-coated flask or plate. 
	&#8203;NOTE: Passaging should be done once a week at a ratio of 1:4.</li>
</ol>
8. Characterization of mitoDendra2-GECs
<ol>
	<li>Perform immunofluorescence staining for CD31 on differentiated cells7,18,19,20 (Figure 4D). Use other markers such as vWF and Isolectin-B4 as well20.</li>
	<li>To confirm mitochondrial specificity of Dendra2, label cells with mitochondrial-specific markers by incubating the cells in growth medium containing 100 nM of mitochondrial tracker for 30 min at 37 &#176;C, 5% CO2.</li>
	<li>Remove the medium and wash the cells gently with pre-warmed medium without phenol red to perform live imaging. 
	NOTE: Alternatively, cells may be fixed using formaldehyde fixatives (4%) for 30 min followed by washing several times in PBS. Fixed cells can be stored at 4 &#176;C in 1x PBS until use. A confirmation with flow cytometry is also possible: label cells with anti-CD31, anti-PECAM, anti-CD45. GECs should stain negative for podocyte markers (synaptopodin, Nphs1, Podxl), mesangial cell markers (PDGFR&#946;, angiotensinogen), and tubular cell markers (Aquaporin, Aminopeptidase-N). These markers serve as negative controls. Due to the mitochondrial-specific Dendra2 expression of these cells, avoid using the FITC channel.</li>
</ol>
9. Live cell imaging of mitochondria structural changes in response to oxidative stress or high glucose
<ol>
	<li>Plate 1 x 105 GECs on a 35 mm glass-bottom dish coated with collagen IV using endothelial cell growth medium (Table of Materials). After 24 h, change to medium without phenol red. Cells have green, fluorescent mitochondria.</li>
	<li>Set the environmental chamber at 37 &#176;C, 5% humidity, and 5% CO2 1 h prior to the start of the experiment.</li>
	<li>Under a confocal microscope, use a 40x/1.2 W objective to adjust the focus and select the region of interest. Incubate the cells with 2mL of 5 mM or 25 mM glucose for 60 min.</li>
	<li>Expose the cells to a 405 nm laser (4% laser power) to photo-convert a subpopulation of mitochondria from green to red (Figure 5A-B, D-E).</li>
	<li>Use the 561 nm laser to excite Dendra2 in the unconverted state to visualize the red fluorescence. See the detailed photo-switching protocol as previously described16. The fusion events appear as yellow fluorescence after several minutes because of green matrix and red matrix fusion (Figure 4C, F).</li>
</ol>

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Authors have no competing financial interests to declare.

Mitochondria are critical for cellular metabolism, homeostasis, and stress responses, and their dysfunction is linked to many diseases, including kidney disease. Mitochondria have a role in the pathologic generation of excessive reactive oxygen species (ROS), the regulation of intracellular calcium levels, cell death pathways, and cytoskeletal dynamics21,22,23.
The isolation of murine GECs is challeng...

The glomerulus is critical for blood filtration by restricting the passage of large molecules through the glomerular filtration barrier1,2. The glomerulus contains four cell types: parietal epithelial cells, podocytes (visceral epithelial cells), glomerular endothelial cells (GEC), and mesangial cells3. The glomerular endothelium is characterized by a unique vascular structure, as per the presence of fenestrae required for large filtration volumes4. The apical surface of the glomerular endothelium is covered with a negatively charged glycocalyx layer and a coat called the endothelial surface layer that creates a space between the endothelium and blood. This structure provides high charge selectivity restricting the passage of negatively charged molecules such as albumin and preventing leukocyte and platelet adhesion5.
GECs are very sensitive to metabolic changes, such as the hyperglycemia associated with the diabetic milieu. Indeed, diabetes leads to increased circulation of noxious substances, the saturation of glucose metabolism pathways, and disturbed cellular redox balance3,6. Moreover, the increase in reactive oxygen species induces mitochondrial dysfunction, which affects endothelial function7.
The overall goal of the current protocol is to isolate immortalized glomerular endothelial cells with fluorescent mitochondrial features. Indeed, the cell culture of primary GECs has a limited proliferative cycle and early senescence8. In addition, the presence of fluorescent mitochondria helps examine fission and fusion events in response to hyperglycemia or any other treatment. As an alternative method, other labs used h-TERT to immortalize cells in vitro9.
The method described here allows for the isolation of conditionally immortalized mitoDendra2 glomerular endothelial cells from 4-6-week-old animals (Figure 1). This detailed protocol describes the use of transgenic mice (H-2Kb-tsA58) harboring the simian virus 40 large tumor antigen (SV40 TAg) gene10,11 for generating thermolabile conditionally immortalized cells. The tsA58 TAg gene product is functional at the permissive temperature of 33 &#176;C under the control of the inducible 5&#39; flanking promoter of the mouse H-2Kb gene, which is increased above basal levels upon exposure to interferon gamma (IFN&#947;), therefore maintaining the conditional proliferation phenotype12. H-2Kb is rapidly degraded at the non-permissive temperature of 37 &#176;C in the absence of IFN&#947;, removing the immortalizing function of the tsA58Tag in cells and allowing the cells to develop a more differentiated phenotype13,14,15. Optional crossing of H-2Kb-tsA58 transgenic mice with PhAM mice, which express a mitochondria-specific (subunit VIII of cytochrome c oxidase) Dendra2-green, allows the live detection of fluorescent mitochondria16. Dendra2 green fluorescence switches to red fluorescence after exposure to a 405 nm laser16. When mitochondria fuse after photo-switching, they form elongated shapes that appear yellow from the exchange of green and yellow material or appear red when they undergo fission7,17. The mitoDendra2-GECs are a great tool for studying the cellular responses of GEC mitochondria to different stimuli.

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		<tr>
			<td>100 &#181;m cell strainer</td>
			<td>Fisher</td>
			<td>22-363-549</td>
			<td></td>
		</tr>
		<tr>
			<td>1ml Insulin Syringes</td>
			<td>BD</td>
			<td>329424</td>
			<td></td>
		</tr>
		<tr>
			<td>25G butterfly</td>
			<td>BD</td>
			<td>367298</td>
			<td></td>
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		<tr>
			<td>3 mm cutting edge scissors</td>
			<td>F.S.T</td>
			<td>15000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>30ml syringe</td>
			<td>BD Biooscience</td>
			<td>309650</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m cell strainer</td>
			<td>Fisher</td>
			<td>22-363-547</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m nylon mesh</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bonn Scissors</td>
			<td>F.S.T</td>
			<td>14184-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Fisher</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31</td>
			<td>abcam</td>
			<td>ab7388</td>
			<td></td>
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			<td>Collagenase type I</td>
			<td>Corning</td>
			<td>354236</td>
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			<td>Collagenase type II</td>
			<td>SIGMA</td>
			<td>C6885</td>
			<td>125CDU/mg</td>
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		<tr>
			<td>Collagene type IV</td>
			<td>SIGMA</td>
			<td>C5533-5M</td>
			<td></td>
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		<tr>
			<td>Dnase-I</td>
			<td>Qiagen</td>
			<td>79254</td>
			<td></td>
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		<tr>
			<td>Dynabeads 450</td>
			<td>Thermofisher Scientific</td>
			<td>14013</td>
			<td></td>
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		<tr>
			<td>endothelial cells growth medium</td>
			<td>Lonza</td>
			<td>cc-3156</td>
			<td></td>
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		<tr>
			<td>Extra fine graefe forceps</td>
			<td>F.S.T</td>
			<td>11150-10</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gemini</td>
			<td>100-106</td>
			<td>Heat inactivated</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Thermofisher</td>
			<td>33016015</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>F.S.T Dumont</td>
			<td>E6511</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>GIBCO</td>
			<td>14065-056</td>
			<td></td>
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		<tr>
			<td>IFNg</td>
			<td>Cell Science</td>
			<td>CRI001B</td>
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			<td>Immortomouse</td>
			<td>Jackson laboratory</td>
			<td>32619</td>
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			<td>L-Glutamine 100x</td>
			<td>Thermofisher Scientific</td>
			<td>25030081</td>
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		<tr>
			<td>Magnetic particle concentrator</td>
			<td>Thermofisher Scientific</td>
			<td>12320D</td>
			<td></td>
		</tr>
		<tr>
			<td>mitotracker</td>
			<td>Thermofisher Scientific</td>
			<td>M7512</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1X</td>
			<td>Corning</td>
			<td>46-013-CM</td>
			<td></td>
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		<tr>
			<td>penecillin streptomycin 100x</td>
			<td>Thermofisher Scientific</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr>
			<td>PhaM mice</td>
			<td>Jackson laboratory</td>
			<td>18397</td>
			<td>B6;129S-Gt(ROSA)26Sortm1.1(CAG-COX8A/Dendra2)Dcc/J</td>
		</tr>
		<tr>
			<td>Protease (10 mg/ml)</td>
			<td>SIGMA</td>
			<td>P6911</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>GIBCO</td>
			<td>3945</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate 100mM</td>
			<td>Thermofisher Scientific</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard pattern forceps</td>
			<td>&#160;F.S.T</td>
			<td>11000-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors - Sharp-Blunt</td>
			<td>F.S.T</td>
			<td>14008-14</td>
			<td></td>
		</tr>
		<tr>
			<td>synaptopodin</td>
			<td>Santa Cruz</td>
			<td>sc-515842</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.05%</td>
			<td>Thermofisher Scientific</td>
			<td>25300054</td>
			<td></td>
		</tr>
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</table>

isolation of conditionally immortalized mouse glomerular endothelial cells with fluorescent mitochondria

Glomerular endothelial cell (GEC) dysfunction can initiate and contribute to glomerular filtration barrier breakdown. Increased mitochondrial oxidative stress has been suggested as a mechanism resulting in GEC dysfunction in the pathogenesis of some glomerular diseases. Historically the isolation of GECs from in vivo models has been notoriously challenging due to difficulties in isolating pure cultures from glomeruli. GECs have complex growth requirements in vitro and a very limited lifespan. Here, we describe the procedure for isolating and culturing conditionally immortalized GECs with fluorescent mitochondria, enabling the tracking of mitochondrial fission and fusion events. GECs were isolated from the kidneys of a double transgenic mouse expressing the thermolabile SV40 TAg (from the Immortomouse), conditionally promoting proliferation and suppressing cell differentiation, and a photo-convertible fluorescent protein (Dendra2) in all mitochondria (from the photo-activatable mitochondria [PhAMexcised] mouse). The stable cell line generated allows for cell differentiation after inactivation of the immortalizing SV40 TAg gene and photo-activation of a subset of mitochondria causing a switch in fluorescence from green to red. The use of mitoDendra2-GECs allows for live imaging of fluorescent mitochondria&#39;s distribution, fusion, and fission events without staining the cells.

In this article, a detailed protocol for the isolation of conditionally immortalized glomerular endothelial cells with stable fluorescent mitochondria (mitoDendra2-GECs) is described (Figure 1). The use of young 6-10-week-old mice is essential to obtain a substantial number of healthy cells. After 3 days of culture, cells start growing slowly from the isolated glomeruli, as shown in Figure 3G. After 7 days, cells are heterogeneous, showing other glomerular cell ...

Watch this Scientific Journal Video about Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria at JoVE.com

Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria

The article describes the method for isolating conditionally immortalized glomerular endothelial cells from the kidneys of transgenic mice expressing the thermolabile simian virus 40 and photo-activatable mitochondria, PhAMexcised. We describe the procedure for glomeruli isolation from whole kidneys using beads, digestion steps, seeding, and culturing of GECs-CD31 positive.

Glomerular endothelial cell (GEC) dysfunction can initiate and contribute to glomerular filtration barrier breakdown. Increased mitochondrial oxidative ...

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2.0K Views.  The Icahn School of Medicine at Mount Sinai. The article describes the method for isolating conditionally immortalized glomerular endothelial cells from the kidneys of transgenic mice expressing the thermolabile simian virus 40 and photo-activatable mitochondria, PhAMexcised. We describe the procedure for glomeruli isolation from whole kidneys using beads, digestion steps, seeding, and culturing of GECs-CD31 positive.

Video: Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps ...

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized near the mitochondria. Support for this possibility is derived from recent studies, in which many mRNAs encoding mitochondrial proteins were shown to be localized to the mitochondria vicinity. Together with earlier demonstrations of ribosomes&#8217; association with the outer membrane, these results suggest a localized translation process. Such localized translation may improve import efficiency, provide unique regulation sites and minimize cases of ectopic expression. Diverse methods have been used to characterize the factors and elements that mediate localized translation. Standard among these is subcellular fractionation by differential centrifugation. This protocol has the advantage of isolation of mRNAs, ribosomes and proteins in a single procedure. These can then be characterized by various molecular and biochemical methods. Furthermore, transcriptomics and proteomics methods can be applied to the resulting material, thereby allow genome-wide insights. The utilization of yeast as a model organism for such studies has the advantages of speed, costs and simplicity. Furthermore, the advanced genetic tools and available deletion strains facilitate verification of candidate factors.

Many mRNAs encoding mitochondrial proteins are associated with the mitochondria outer membrane. We describe a subcellular fractionation procedure aimed at isolation of yeast mitochondria with its associated mRNAs and ribosomes. This protocol can be applied to cells grown under diverse conditions&#160;in order to reveal mechanisms of mRNA localization and localized translation near the mitochondria.

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...