Name: methods to assess beta cell death mediated by cytotoxic t lymphocytes
Uploaded: 2011-06-16T12:00:00
Description: Watch this Scientific Journal Video about Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes at JoVE.com

इस काम DK074656 स्वास्थ्य और AI56374 (CEM) के राष्ट्रीय संस्थानों से अनुदान के रूप में के रूप में अच्छी तरह से किशोर मधुमेह अनुसंधान फाउंडेशन द्वारा समर्थित किया गया.

1. कक्षों की तैयारी <ol><li> लक्ष्य सेल संस्कृति: संस्कृति, माउस बीटा सेल लाइनों, एनआईटी-1 और एनआईटी-4 DMEM में एक पहले से वर्णित 1,2 10% FBS, 0.02% BSA, गैर आवश्यक एमिनो एसिड, 15mm HEPES, 16.5mM के साथ पूरक था Glocose और पेनिसिलीन / स्ट्रेप्...

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साइटोटोक्सिक टी सेल की मध्यस्थता बीटा कोशिका मृत्यु 5 T1D के मुख्य pathophysiology है. CML के 51 करोड़ रिलीज का उपयोग assays हमें effector लक्ष्य 6 प्रतिक्रिया की डिग्री का अध्ययन करने के लिए अनुमति देते हैं. हालांकि...

<table border="1"><tbody><tr><th> अभिकर्मक के नाम </th><th> कंपनी </th><th> सूचीपत्र संख्या </th><th> टिप्पणियां (वैकल्पिक) </th></tr><tr><td> CR-51 </td><td> Perkin एल्मर </td><td> NEZ030500IMC </td><td></td></tr><tr><td> पीबीएस </td><td> Cellgro </td><td> 21-040-60 </td><td></td></tr><tr><td> Tetramethyl rhodamine मिथाइल एस्टर (TMRM) </td><td> Invitrogen </td><td> T668 </td><td></td></tr><tr><td> Picogreen </td><td> Invitrogen </td><td> P7581 </td><td></td></tr><tr><td> AI4 mimotope </td><td> mimotopes.com </td><td> अमीनो एसिड अनुक्रम: YFIENYLEL </td><td></td></tr><tr><td> आईएल -2 </td><td> अनुसंधान एवं विकास </td><td> 485 एमआई </td><td></td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11885 </td><td></td></tr><tr><td> FBS </td><td> HyClone </td><td> SH30071.03 </td><td></td></tr><tr><td> गोजातीय सीरम albumin </td><td> सिग्मा </td><td> A8412 </td><td></td></tr><tr><td> HEPES </td><td> Cellgro </td><td> 25-060-सीएल </td><td></td></tr><tr><td> गैर आवश्यक अमीनो एसिड के सदस्य </td><td> GIBCO </td><td> 11140 </td><td></td></tr><tr><td> पेनिसिलीन / स्ट्रेप्टोमाइसिन </td><td> मिथुन राशि </td><td> 400-109 </td><td></td></tr><tr><td> Phenol लाल मुक्त DMEM </td><td> सिग्मा </td><td> D5303 </td><td></td></tr><tr><td> सीओ 2 इनक्यूबेटर </td><td> थर्मो फिशर </td><td> HeraCell 150i </td><td> सेल संस्कृति के लिए बाँझ रखा </td></tr><tr><td> जैविक सुरक्षा कैबिनेट </td><td> थर्मो फिशर </td><td> 1440 Forma </td><td></td></tr><tr><td> प्रयोज्य संस्कृति ट्यूबों, 6x50 मिमी निम्बुड़ा ग्लास </td><td> मछुआ </td><td> 14 958 - ए </td><td></td></tr><tr><td> गामा काउंटर </td><td> Perkin एल्मर </td><td> विज़ार्ड स्वचालित 1470 गामा काउंटर </td><td></td></tr><tr><td> Confocal खुर्दबीन </td><td> Zeiss </td><td> LSM 510 मेटा Confocal </td><td> Motorized मंच और सेल रहते कक्ष </td></tr><tr><td> (1ml, 200μl, 20μl, 10μl, 2μl) पिपेट </td><td> Eppendorf </td><td></td><td></td></tr><tr><td> ट्यूबें (एम्बर) </td><td> मछुआ </td><td> 50819772 </td><td></td></tr><tr><td> अपकेंद्रित्र </td><td> Sorvall लीजेंड आरटी + </td><td> 75004377 </td><td></td></tr><tr><td> Hemacytometer </td><td> फिशर साइंटिफिक </td><td> 267110 </td><td></td></tr><tr><td> ईमानदार माइक्रोस्कोप </td><td> Zeiss </td><td> Axioskop40 </td><td></td></tr><tr><td> NOD.Cg Rag1 tm1Mom (TcraAI4) टीजी 1Dvs/DvsJ चूहे </td><td> जैक्सन प्रयोगशाला </td><td> 004347 </td><td></td></tr><tr><td> NOD.Cg Rag1 tm1Mom (TcrbAI4) टीजी 1Dvs/DvsJ चूहे </td><td> जैक्सन प्रयोगशाला </td><td> 004348 </td><td></td></tr><tr><td> इशारा - AI4α / β F1 चूहों </td><td> N / A </td><td> N / A </td><td> UF जानवर सुविधा में नस्ल </td></tr></tbody></table>

methods to assess beta cell death mediated by cytotoxic t lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Watch this Scientific Journal Video about Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes at JoVE.com

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

सेल की मध्यस्थता lymphocytotoxicity assays (CML) इन विट्रो में और autoreactive प्रतिक्रियाओं कोशिका मृत्यु का अध्ययन तंत्र का परीक्षण करने के लिए इस्तेमाल किया जा सकता है. हालांकि, रहने कक्ष फ्लोरोसेंट रंजक, प्रकार और सेल के रूप में के रूप में अच्छी तरह से मौत उपयोग रास्ते के कैनेटीक्स के साथ confocal सूक्ष्म इमेजिंग तकनीक का उपयोग कर अधिक से अधिक विस्तार में अध्ययन किया जा सकता है.

साइटोटोक्सिक टी lymphocytes द्वारा बीटा सेल मौत मध्यस्थता का आकलन करने के तरीके

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

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