Summary

Методы оценки бета опосредованная гибель клеток от цитотоксических Т-лимфоцитов

Published: June 16, 2011
doi:

Summary

Клеточный lymphocytotoxicity (ХМЛ), анализы могут быть использованы для тестирования аутореактивных ответы и изучение механизмов смерти клеток в пробирке. Тем не менее, с использованием живых-клеточной конфокальной микроскопии изображений с флуоресцентными красителями, типа и кинетики гибели клеток, а также путей использованы может быть изучен более детально.

Abstract

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°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.

Protocol

1. Подготовка клетки Культуры клеток-мишеней: Культура мыши бета клеточных линий, NIT-1 и NIT-4, было описано выше 1,2 в DMEM с добавлением 10% FBS, 0,02% BSA, Номера для незаменимая аминокислота, 15 мм HEPES, 16.5мм Glocose и пенициллина / стрептомицина. Для 51 Cr маркировки, семенами клеток в плоск?…

Discussion

Цитотоксических Т-клеток опосредованной смерти бета-клеток является основным патофизиологии T1D 5. CML анализов использованием 51 Cr релизе позволяют исследовать степень эффекторных-целевой отклик 6. Тем не менее, детальный процесс и пути Т клеточного гибели клеток бета-п…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Эта работа была поддержана грантами от Национального института здоровья и DK074656 AI56374 (CEM), а также несовершеннолетних исследовательского фонда диабета.

Materials

Name of the reagent Company Catalogue number Comments (optional)
Cr-51 Perkin Elmer NEZ030500IMC  
PBS Cellgro 21-040-60  
Tetramethyl Rhodamine Methyl Ester (TMRM) Invitrogen T668  
Picogreen Invitrogen P7581  
AI4 mimotope mimotopes.com amino acid sequence: YFIENYLEL  
IL-2 R & D 485-MI  
DMEM Invitrogen 11885  
FBS HyClone SH30071.03  
Bovine Serum Albumin Sigma A8412  
HEPES Cellgro 25-060-Cl  
MEM Non-Essential Amino Acids GIBCO 11140  
Penicillin/Streptomycin Gemini 400-109  
Phenol red-free DMEM Sigma D5303  
CO2 Incubator Thermo Fisher HeraCell 150i Kept sterile for cell culture
Biological safety cabinet Thermo Fisher Forma 1440  
Disposable culture tubes, 6×50 mm Lime Glass Fisher 14-958-A  
Gamma Counter Perkin Elmer Wizard 1470 Automatic Gamma Counter  
Confocal microscope Zeiss LSM 510 Meta Confocal With motorized stage and live cell chamber
Pipette (1ml, 200μl, 20μl,10μl, 2μl) Eppendorf    
Tubes (Amber) Fisher 50819772  
Centrifuge Sorvall Legend RT+ 75004377  
Hemacytometer Fisher Scientific 267110  
Upright Microscope Zeiss Axioskop40  
NOD.Cg-Rag1tm1Mom Tg(TcraAI4)1Dvs/DvsJ Mice The Jackson Laboratory 004347  
NOD.Cg-Rag1tm1Mom Tg(TcrbAI4)1Dvs/DvsJ Mice The Jackson Laboratory 004348  
NOD-AI4α/ β F1 mice N/A N/A Bred in UF animal facility

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Cite This Article
Chen, J., Grieshaber, S., Mathews, C. E. Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes. J. Vis. Exp. (52), e2724, doi:10.3791/2724 (2011).

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