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 challenging because of their small number, size, matrix factors, and interdependent nature with other glomerular cells that are essential for their survival. The current article presents a detailed protocol for mouse GEC isolation. Specifically, we used transgenic mice as a tool to obtain conditionally immortalized cell lines with high proliferation potential. The transgenic mice, expressing simian virus 40 (SV40) large T antigen tsA58, enable the activation of continuous proliferation under permissive conditions at 33 &#176;C and were here crossed with the PHAMexcised mice expressing mitochondrial-specific fluorescence. The double transgenic mice offered an excellent tool for studying mitochondria structure. Indeed, PHAMexcised mice bear mitochondrial-specific (subunit VIII of cytochrome c oxidase) Dendra2 green, allowing the live detection of fluorescent mitochondria and the observation of fission and fusion events through green-red photo-switching16.
The protocol consists of three significant steps: extracting glomeruli using beads, the proliferation of glomerular cells, and the subsequent purification of GEC using CD31 beads once the cells are in culture. The use of only one digestion with collagenase is sufficient to isolate glomerular cells. Other protocols make use of a second digestion with DNAse and proteinase24. However, we have noticed better cell yields with only one digestion step. The DNAse may remain active and slow down the proliferation of the cells. Furthermore, GEC could be isolated using differential sieving as previously described20, which is not always easy when using mice kidneys.
It is crucial to autoclave all the surgical tools before starting the isolation of the kidneys and the glomeruli to avoid contamination. Moreover, it is important to filter and supplement the solutions with antibiotics to eliminate any source of contaminants. Indeed, mycoplasma infections may cause cytopathology that consequently interferes with every parameter measured in cell culture25.
The isolation of glomeruli relies on the intra-cardiac perfusion of beads. Certainly, slow perfusion with 20 mL of 1x HBSS-containing beads is essential to harvest a high number of glomeruli. The beads will be trapped in the glomeruli, which eases their purification using a magnetic concentrator. The beads might be prepared and stored at 4 &#176;C for up to 15 days before using them to ease the experimental procedure.
Furthermore, digesting glomeruli with continuous shaking is crucial for dissociating a good number of glomeruli from the kidney. On the other hand, the isolation of murine GECs is challenging because of their small size and the presence of other glomerular cells. Therefore, the stepwise isolation of glomeruli, the growth of all glomerular cells in culture, and the subsequent use of CD31-coated beads to purify endothelial cells are vital, specifically for obtaining more significant numbers of viable and conditionally proliferative GECs. Nevertheless, validating the purity of cells with immunostaining and flow cytometry is needed.
In addition, the protocol describes the characterization of the GEC phenotype and the mitochondria fusion/fission assay in vitro. The mitoDendra2-GECs are an excellent tool for studying the cellular responses of GEC mitochondria to different stimuli in vitro without the need for transfection or staining.

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.

<table><tbody><tr><td>100 &amp;micro;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></tr><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 &amp;micro;m cell strainer</td><td>Fisher</td><td>22-363-547</td><td></td></tr><tr><td>40 &amp;micro;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></tr><tr><td>Collagenase type I</td><td>Corning</td><td>354236</td><td></td></tr><tr><td>Collagenase type II</td><td>SIGMA</td><td>C6885</td><td>125CDU/mg</td></tr><tr><td>Collagene type IV</td><td>SIGMA</td><td>C5533-5M</td><td></td></tr><tr><td>Dnase-I</td><td>Qiagen</td><td>79254</td><td></td></tr><tr><td>Dynabeads 450</td><td>Thermofisher Scientific</td><td>14013</td><td></td></tr><tr><td>endothelial cells growth medium</td><td>Lonza</td><td>cc-3156</td><td></td></tr><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></tr><tr><td>IFNg</td><td>Cell Science</td><td>CRI001B</td><td></td></tr><tr><td>Immortomouse</td><td>Jackson laboratory</td><td>32619</td><td>Tg(H2-K1-tsA58)6Kio/LicrmJ</td></tr><tr><td>L-Glutamine 100x</td><td>Thermofisher Scientific</td><td>25030081</td><td></td></tr><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></tr><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>&amp;nbsp;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></tbody></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 types, such as podocytes, parietal epithelial cells, and mesangial cells (Figure 4A). After reaching 70% to 80% confluency, other glomerular cells were removed using a positive selection of endothelial cells with magnetic CD31 labeled beads (Figure 4B-C). The mitoDendra2-GECs expressed CD31 (Figure 4D) and were negative for podocyte marker as shown by synaptopodin negative staining (Figure 4E-F).
The fluorescent mitochondrial feature in the mitoDendra2-GECs allows for studying mitochondria morphology under various conditions in vitro. Here, we tested the effect of high glucose (25mM) on the mitochondria structure of mitoDendra2-GECs (Figure 5D-F). Compared to the elongated mitochondria visible in cells under normal glucose (5mM; Figure 5A-C), high glucose-induced fragmentation or fission of mitochondria as observed by prominent spheroid-shaped mitochondria (Figure 5E). Furthermore, we used a 405 nm laser to photo-convert a selected subpopulation of mitochondria in a single live mitoDendra2-GEC (Figure 5A,D). In Figure 5B and Figure 5E, successful photo-switching of mitochondria from green (488nm) to red (561 nm) in the selected area is presented. Importantly, it allowed for witnessing the fusion events in mitoDendra2-GECs grown under normal glucose, as can be observed by the yellow merged green and red fluorescence as a result of mitochondria matrix fusion (Figure 5C). In contrast, in the high glucose-treated mitoDendra2-GEC, the mitochondria were fragmented and were mainly red (Figure 5F), suggesting that, in the time frame tested, the fission/fusion events were delayed or inhibited due to the damage caused by the high glucose treatment, and this is consistent with previous reports18,19.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64147/64147fig01.jpg" /> 
Figure 1: Representative description of murine GEC isolation. The diagram represents the significant steps of GEC isolation, starting from perfusing mice heart with magnetic beads to isolate the kidney glomeruli, the digestion of collagenase, the culture of glomerular cells, and finally, the purification of GEC using CD31-coated beads. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64147/64147fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64147/64147fig02.jpg" /> 
Figure 2: Description of kidney dissection. Representative image of all the sterile surgical instruments needed for the dissection. Scissors (a) are needed to open the skin of the mouse, and a separate pair of scissors (b,c) are needed to isolate the kidney. Tweezers (d) and (e) are needed to hold the skin. Tweezer (f) is needed to collect the dissected kidney. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64147/64147fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64147/64147fig03.jpg" /> 
Figure 3: Description of glomeruli harvesting. (A) Perfuse the left ventricle with the HBSS-beads solution using a 20 mL syringe. Perforate the inferior vena cava with a 19 G needle. (B) After perfusion, remove the fat from the kidney and cut it into 1 mm pieces. (C) Digest the tissue with collagenase type II. (D) After digestion and centrifugation, three layers are formed; the beads bearing the glomeruli are in the middle layer, as indicated by the black arrow. (E) Separate the isolated beads bearing the glomeruli using the magnetic concentrator. (F) Representative image of freshly isolated glomeruli. (G) Growing cells from isolated glomeruli after 3 days. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64147/64147fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64147/64147fig04.jpg" /> 
Figure 4: Mitochondrial assay in vitro. (A) Glomerular cells after 3 days of culture. (B) GEC after 7 days. (D) Purification of GEC with CD31-labeled beads. Microscopic imaging of CD31 staining. (E) GECs are negative for synaptopodin. (F) Podocytes stained with synaptopodin (488 nm, green fluorescence) as a negative control. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64147/64147fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64147/64147fig05.jpg" /> 
Figure 5: Microscopic imaging of fluorescent mitochondrial fusion in vitro. Images were acquired with a confocal microscope using a 40x water objective. (A-B) Selected area (red circle) was illuminated with a 405 nm line (4% laser power) for 300 bleaching iterations at a speed of 6.3-12.61/pixel, as previously described16. (A-C) Cells in growth media containing 5 mM glucose; green fluorescence (488 nm) shows healthy mitochondria. (B,E) Mitochondria were photo-switched into red (567 nm). (C) Yellow fluorescence detects an active fusion event of GEC treated with 5mM glucose. (D) Cells were treated with high glucose 25 mM for 30 min. Green fluorescence shows fragmented mitochondria and (E) a portion that was photo-switched into red. (F) The figure shows a decreased fusion event as shown by reduced yellow fluorescence. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64147/64147fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

isolation-conditionally-immortalized-mouse-glomerular-endothelial

Nanyang Technological University

Research

JoVE Journal

Biology

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

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.
Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the F&ouml;rster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The ...

Neuroscience

Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies revealing novel paradigms in vascular biology. Applying these methods to models of vascular development requires the optimization of cell isolation techniques from embryonic and postnatal tissues. Cell yield, viability, and purity all need to be maximal to obtain accurate and reproducible results from next-generation sequencing approaches. The neonatal mouse retinal vascularization model is used by researchers to study mechanisms of vascular development. Researchers have used this model to investigate mechanisms of angiogenesis and arterial-venous fate specification during blood vessel formation and maturation. Applying next-generation sequencing techniques to study the retinal vascular development model requires optimization of a method for the isolation of retinal endothelial cells that maximizes cell yield, viability, and purity. This protocol describes a method for murine retinal tissue isolation, digestion, and purification using fluorescence-activated cell sorting (FACS). The results indicate that the FACS-purified CD31+/CD45- endothelial cell population is highly enriched for endothelial cell gene expression and exhibits no change in viability for 60 min post-FACS. Included are representative results of next-generation sequencing approaches on endothelial cells isolated using this method, including bulk RNA sequencing and single-cell RNA sequencing, demonstrating that this method for retinal endothelial cell isolation is compatible with next-generation sequencing applications. This method of retinal endothelial cell isolation will allow for advanced sequencing techniques to reveal novel mechanisms of vascular development.

This protocol describes a method for the isolation of murine postnatal retinal endothelial cells optimized for cell yield, purity, and viability. These cells are suitable for next-generation sequencing approaches.

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies ...

Developmental Biology

Isolation of Primary Mouse Lung Endothelial Cells

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in medical conditions including diabetes, atherosclerosis, sepsis, and acute lung injury. A reliable and reproducible method to isolate pure endothelial cells from mice is needed to investigate the role of endothelial cells in the pathogenesis of these and other conditions. In this protocol, lung microvascular endothelial cells were prepared from 5-7 day old neonatal mouse pups. Lungs are harvested, minced, and enzymatically digested with collagenase I, and released cells are cultured overnight. Endothelial cells are then selected using anti-PECAM1 (CD-31) IgG conjugated to magnetic beads, and cells again are cultured to confluence. A secondary cell selection then occurs with anti-ICAM2 (CD-102) IgG conjugated to magnetic beads to increase the purity of the endothelial cells, and the cells again are cultured to confluence. The entire process takes approximately 7-10 days before the cells can be used for experimentation. This simple protocol yields highly pure (purity &#62;92%) endothelial cells that can be immediately used for in vitro studies, including the studies focused on endothelial cytokine and chemokine production, leukocyte-endothelial interactions, endothelial coagulation pathways, and endothelial permeability. With many knockouts and transgenic mouse lines available, this procedure also lends itself to understanding the function of specific genes expressed by endothelial cells in healthy and pathologic responses to injury, infection, and inflammation.

In this article, primary lung endothelial cells were isolated and cultured from neonatal mice.

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in ...

Immunology and Infection

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 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 Murine Intestinal Mesenchyme Resulting in a High Yield of Telocytes

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

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

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

Medicine

Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina propria. EGCs play important roles in gut homeostasis through the release of various trophic factors and contribute to the integrity of the epithelial barrier. Most studies of primary enteric glial cultures use cells isolated from the myenteric plexus after enzymatic dissociation. Here, a non-enzymatic method to isolate and culture EGCs from the intestinal submucosa and lamina propria is described. After manual removal of the longitudinal muscle layer, EGCs were liberated from the lamina propria and submucosa using sequential HEPES-buffered EDTA incubations followed by incubation in commercially available non-enzymatic cell recovery solution. The EDTA incubations were sufficient to strip most of the epithelial mucosa from the lamina propria, allowing the cell recovery solution to liberate the submucosal EGCs. Any residual lamina propria and smooth muscle was discarded along with the myenteric glia. EGCs were easily identified by their ability to express glial fibrillary acidic protein (GFAP). Only about 50% of the cell suspension contained GFAP+ cells after completing tissue incubations and prior to plating on the poly-D-lysine/laminin substrate. However, after 3 days of culturing the cells in glial cell-derived neurotrophic factor (GDNF)-containing culture media, the cell population adhering to the substrate-coated plates comprised of &#62;95% enteric glia. We created a hybrid mouse line by breeding a hGFAP-Cre mouse to the ROSA-tdTomato reporter line to track the percentage of GFAP+ cells using endogenous cell fluorescence. Thus, non-myenteric enteric glia can be isolated by non-enzymatic methods and cultured for at least 5 days.

Here, we describe the isolation of enteric-glial cells from the intestinal-submucosa using sequential EDTA incubations to chelate divalent cations and then incubation in non-enzymatic cell recovery solution. Plating the resultant cell suspension on poly-D-lysine and laminin results in a highly enriched culture of submucosal glial cells for functional analysis.

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina ...

Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). Therefore, assessing mitochondrial function in primary TECs may provide valuable insight into the bioenergetic status of the cells, providing insight into the pathophysiology of CKD. While there are a number of complex protocols available for the isolation and purification of proximal tubules in different species, the field lacks a cost-effective method optimized for&#160;tubular cell isolation without the need for purification. Here, we provide an isolation protocol that allows for studies focusing on both primary mouse proximal and distal renal TECs. In addition to cost-effective reagents and minimal animal procedures required in this protocol, the isolated cells maintain high energy levels after isolation and can be sub-cultured up to four&#160;passages, allowing for continuous studies. Furthermore, using a high throughput extracellular flux analyzer, we assess the mitochondrial respiration directly in the isolated TECs in a 96-well plate for which we provide recommendations for the optimization of cell density and compound concentration. These observations suggest that this protocol can be used for renal tubular ex vivo studies with a consistent, well-standardized production of renal TECs. This protocol may have broader future applications to study mitochondrial dysfunction associated with renal disorders for drug discovery or drug characterization purposes.

This protocol provides a cost-effective approach to isolate and characterize mouse primary renal tubular cells that can subsequently be sub-cultured to assess renal biological functions ex vivo, including mitochondrial bioenergetics.

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). ...

Isolation of Primary Mouse Retinal Glial M&#252;ller Cells

The primary supporting cell of the retina is the retinal glial M&#252;ller cell. They cover the entire retinal surface and are in close proximity to both the retinal blood vessels and the retinal neurons. Because of their growth, M&#252;ller cells perform several crucial tasks in a healthy retina, including the uptake and recycling of neurotransmitters, retinoic acid compounds, and ions (like potassium K+). In addition to regulating blood flow and maintaining the blood-retinal barrier, they also regulate the metabolism and the supply of nutrients to the retina. An established procedure for isolating primary mouse M&#252;ller cells is presented in this manuscript. To better understand the underlying molecular processes involved in the various mouse models of ocular disorders, M&#252;ller cell isolation is an excellent approach. This manuscript outlines a detailed procedure for M&#252;ller cell isolation from mice. From enucleation to seeding, the entire process lasts about a few hours. For 5-7 days after seeding, the media shouldn&#39;t be changed in order to allow the isolated cells to grow unhindered. Cell characterization using morphology and distinct immunofluorescent markers comes next in the process. Maximum passages for cells are 3-4 times.

This manuscript describes a detailed protocol for isolating retinal glial M&#252;ller cells from mouse eyes. The protocol starts with enucleation and dissection of mouse eyes, followed by isolation, seeding, and culturing of M&#252;ller cells.

The primary supporting cell of the retina is the retinal glial M&#252;ller cell. They cover the entire retinal surface and are in close proximity to both ...

Optimizing Isolation and Purification of Murine Glomerular Mesangial Cells

Mesangial cells (MCs) are stromal cells located in the middle space of the glomerulus, with pivotal functions in glomerular homeostasis. Methods for isolating, purifying, and culturing glomerular MCs have been developed and optimized since the 1980s for use in biomedical research, particularly in the field of nephrology. Mice are the most frequently used experimental animal models in research on renal diseases. In this study, we developed an optimized protocol for murine MCs isolation and ex vivo cell culture. These cells can be passaged multiple times, frozen, revived, and cultured without compromising cell growth or protein expression. This optimized approach significantly reduces the study duration for researchers and enables long-term cell preservation. The necessary equipment for the procedures is easily accessible in a basic biomedical laboratory, and the procedural steps are straightforward. The acquisition of target cells requires only 2-3 weeks, a reduction of at least 1 week compared to existing methods.

This study developed an optimized protocol for the isolation of murine mesangial cells (MCs) and their ex vivo cell culture. These cells can be passaged multiple times, frozen, revived, and cultured without compromising cell growth or protein expression.

Mesangial cells (MCs) are stromal cells located in the middle space of the glomerulus, with pivotal functions in glomerular homeostasis. Methods for ...

Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria

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Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells

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Optimizing Isolation and Purification of Murine Glomerular Mesangial Cells