Name: assessment of mitochondrial functions and cell viability in renal cells overexpressing protein kinase c isozymes
Uploaded: 2013-01-07T12:00:00
Description: Watch this Scientific Journal Video about Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes at JoVE.com

This work was supported by a grant from the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, 2R01DK59558 (to G.N.). UAMS Translational Research Institute supported by the National Institutes of Health National Center for Research Resources grant UL1 RR029884 provided partial funding for Flow Cytometry Core at UAMS. We thank Dr. Peipei Ping (University of California at Los Angeles; Los Angeles, CA) for providing an aliquot of adenovirus carrying cDNA coding the dominant negative (inactive) mutant of PKC-&epsilon; and Dr. Allen Samarel (Loyola University Medical Center; Maywood, IL) for providing an aliquot of adenoviral vector coding the constitutively active mutant of PKC-&epsilon;. We also thank Drs. Peter Parker and Peter Sugden (Imperial College London, London, UK) for providing cDNA coding constitutively active PKC-&epsilon;.

1. प्राथमिक संस्कृति के लिए गुर्दे समीपस्थ नलिकाओं का अलगाव <ol><li> खरगोश, आबकारी anesthetize दोनों गुर्दे और उन्हें एक लामिना का प्रवाह हुड में बाँझ प्रस्तुत करने का बफर (DMEM/F12 मध्यम) के साथ भरा एक पेट्री डिश में जगह...

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यहाँ प्रस्तुत दृष्टिकोण गुर्दे समीपस्थ ट्यूबलर कोशिकाओं के प्राथमिक संस्कृति में पीकेसी के व्यक्तिगत isozymes की overexpression के लिए अनुमति देता है. इस दृष्टिकोण के कई शक्तियों हैं: 1. यह (गुर्दे समीपस्थ ट्यूबलर क...

<table border="1"><tbody><tr><td> अभिकर्मक के नाम </td><td> कंपनी </td><td> सूचीपत्र संख्या </td></tr><tr><td> लामिना का प्रवाह हुड </td><td> थर्मो इलेक्ट्रॉन निगम </td><td> 1104 प्रपत्र </td></tr><tr><td> 2 मिलीग्राम और 15 मिलीग्राम Dounce ऊतक चक्की </td><td> व्हिटन </td><td> ३,५७,५४४ 989-24,607 </td></tr><tr><td> 85 और 235 माइक्रोन नायलॉन जाल </td><td> छोटे भागों </td><td> CMN - 0085 - 10YD CMN - 0250 - 10YD </td></tr><tr><td> 50 मिलीलीटर बाँझ अपकेंद्रित्र ट्यूब </td><td> BIOLOGIX </td><td> BCT - पी 50BS </td></tr><tr><td> 1.5 मिलीलीटर सूक्ष्म ट्यूब </td><td> Sarstedt </td><td> 72.690.001 </td></tr><tr><td> 35 x 10 मिमी बाँझ संस्कृति व्यंजन </td><td> कोर्निंग </td><td> 430165 </td></tr><tr><td> Jouan अपकेंद्रित्र </td><td> Jouan </td><td> Jouan CR3 11175704 रोटार: Jouan T40 </td></tr><tr&gt; <td> समायोज्य अपकेंद्रित्र सूक्ष्म </td><td> सिग्मा </td><td> मॉडल 1 - 15 </td></tr><tr><td> जैविक ऑक्सीजन की निगरानी </td><td> YSI शामिल </td><td> YSI मॉडल 5300A </td></tr><tr><td> एकल कक्ष माइक्रो ऑक्सीजन प्रणाली </td><td> YSI शामिल </td><td> 5356S </td></tr><tr><td> ऑक्सीजन जांच </td><td> YSI शामिल </td><td> 5331A </td></tr><tr><td> स्नान परिसंचारी </td><td> YSI शामिल </td><td> 5310 </td></tr><tr><td> KCl और मानक झिल्ली किट </td><td> YSI शामिल </td><td> 5775 </td></tr><tr><td> चुंबकीय दोषी </td><td> YSI शामिल </td><td> 5222 </td></tr><tr><td> Flatbed रिकॉर्डर </td><td> Kipp और ZONEN </td><td> BD 11e </td></tr><tr><td> 48-अच्छी तरह से और 96-अच्छी तरह से पारदर्शी प्लेटें </td><td> Costar </td><td> 3679 3548, </td></tr><tr><td> Thermomixer आर </td><td> एppendorf </td><td> 5355 21919 </td></tr><tr><td> कक्षीय हिलनेवाला MAXQ 2000 </td><td> थर्मो वैज्ञानिक </td><td> 2000 SHKA </td></tr><tr><td> स्पेक्ट्रा FLUOR प्लस (absorbance / प्रतिदीप्ति / luminescence पाठक) </td><td> Tecan </td><td> F129005 </td></tr><tr><td> जल तख्ताबंदीवाला अमेरिका Autoflow स्वचालित सीओ 2 इनक्यूबेटर </td><td> NUAIRE </td><td> NU 4850 </td></tr><tr><td> 12x75 मिमी प्रवाह cytometry के लिए polystyrene संस्कृति टेस्ट ट्यूब </td><td> फिशर ब्रांड </td><td> 14-961-20 </td></tr><tr><td> Axioskop पानी विसर्जन उद्देश्य 63x / 0,90 डब्ल्यू </td><td> कार्ल Zeiss </td><td> 114846 00 67 44 ACHROPLAN </td></tr><tr><td> / DMEM F12 </td><td> Cellgro </td><td> 99 - 830 - पीबी </td></tr><tr><td> DMEM / F12 हाम </td><td> सिग्मा </td><td> डी 2906 - 1L </td></tr><tr><td> Deferoxamine Mesylate </td><td> हॉस्पिटलra </td><td> D110 </td></tr><tr><td> Collagenase प्रकार मैं </td><td> वर्थिंगटन </td><td> 4196 </td></tr><tr><td> Trypsin अवरोध करनेवाला </td><td> सिग्मा </td><td> 6522 टी - 500mg </td></tr><tr><td> 5,5, 6,6 &#39;tetrachloro-1, 1, 3,3&#39; tetraethylbenzimidazolylcarbocyanine (जे.सी. 1) आयोडाइड </td><td> Invitrogen </td><td> T3168 </td></tr><tr><td> Mitotracker लाल </td><td> Invitrogen </td><td> M22425 </td></tr><tr><td> एटीपी Bioluminescence परख किट एच एस द्वितीय </td><td> Roche </td><td> 11 709 001 699 </td></tr><tr><td> FITC समाधान annexin वी </td><td> BioVision </td><td> 1001 - 200 </td></tr><tr><td> प्रवाह cytometer </td><td> बी.डी. बायोसाइंसेज </td><td> BD FACSCalibur </td></tr></tbody></table>

assessment of mitochondrial functions and cell viability in renal cells overexpressing protein kinase c isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

चित्रा 1 से पता चलता है कि adenoviral वितरण सीडीएनए constitutively (caPKC ε) सक्रिय और निष्क्रिय (dnPKC ε) काफी वृद्धि हुई PKC ε की RPTC और mitochondria में प्रोटीन के स्तर में PKC ε परिणामों के म्यूटेंट कोडन. वेक्टर caPKC - ε सीडीएनए ले जाने के स...

Watch this Scientific Journal Video about Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes at JoVE.com

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

प्रोटीन kinase सी (PKC) mitochondrial श्वसन और oxidative phosphorylation और सेल व्यवहार्यता पर जुड़े कार्यों पर isozymes के सक्रियण के प्रभाव का वर्णन कर रहे हैं. दृष्टिकोण adenoviral चुनिंदा प्राथमिक सेल संस्कृति और assays की एक किस्म में PKC isozymes overexpress mitochondrial कार्यों और सेल की ऊर्जा स्थिति का निर्धारण तकनीक adapts.

Mitochondrial गुर्दे कोशिकाओं overexpressing प्रोटीन kinase सी Isozymes में और कार्य सेल व्यवहार्यता का आकलन

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

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