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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 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's distribution, fusion, and fission events without staining the cells.
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 c....
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
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 .......
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.......
Authors have no competing financial interests to declare.
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.
....Name | Company | Catalog Number | Comments |
100 µm cell strainer | Fisher | 22-363-549 | |
1ml Insulin Syringes | BD | 329424 | |
25G butterfly | BD | 367298 | |
3 mm cutting edge scissors | F.S.T | 15000-00 | |
30ml syringe | BD Biooscience | 309650 | |
40 µm cell strainer | Fisher | 22-363-547 | |
40 µm nylon mesh | |||
Bonn Scissors | F.S.T | 14184-09 | |
Bovine serum albumin | Fisher | BP1600-100 | |
CD31 | abcam | ab7388 | |
Collagenase type I | Corning | 354236 | |
Collagenase type II | SIGMA | C6885 | 125CDU/mg |
Collagene type IV | SIGMA | C5533-5M | |
Dnase-I | Qiagen | 79254 | |
Dynabeads 450 | Thermofisher Scientific | 14013 | |
endothelial cells growth medium | Lonza | cc-3156 | |
Extra fine graefe forceps | F.S.T | 11150-10 | |
FBS | Gemini | 100-106 | Heat inactivated |
Fibronectin | Thermofisher | 33016015 | |
Fine forceps | F.S.T Dumont | E6511 | |
HBSS | GIBCO | 14065-056 | |
IFNg | Cell Science | CRI001B | |
Immortomouse | Jackson laboratory | 32619 | Tg(H2-K1-tsA58)6Kio/LicrmJ |
L-Glutamine 100x | Thermofisher Scientific | 25030081 | |
Magnetic particle concentrator | Thermofisher Scientific | 12320D | |
mitotracker | Thermofisher Scientific | M7512 | |
PBS 1X | Corning | 46-013-CM | |
penecillin streptomycin 100x | Thermofisher Scientific | 10378016 | |
PhaM mice | Jackson laboratory | 18397 | B6;129S-Gt(ROSA)26Sortm1.1(CAG-COX8A/Dendra2)Dcc/J |
Protease (10 mg/ml) | SIGMA | P6911 | |
RPMI | GIBCO | 3945 | |
Sodium Pyruvate 100mM | Thermofisher Scientific | 11360070 | |
Standard pattern forceps | F.S.T | 11000-12 | |
Surgical Scissors - Sharp-Blunt | F.S.T | 14008-14 | |
synaptopodin | Santa Cruz | sc-515842 | |
Trypsin 0.05% | Thermofisher Scientific | 25300054 |
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