This work was supported by grants from the National Institutes of Health DK074656 and AI56374 (CEM), as well as the Juvenile Diabetes Research Foundation.

1.	Preparation of cells
<ol>
	<li>Target cell culture: Culture of the mouse beta cell lines, NIT-1 and NIT-4, was a previously described1,2 in DMEM supplemented with 10% FBS, 0.02%BSA, Non-essential Amino Acid, 15mM HEPES, 16.5mM Glocose and Penicillin/Streptomycin. For 51Cr labeling, seed cells in a flat-bottom 96-well culture plate at 5X104 cells/well in 200 &mu;l culture medium. For microscopy experiments, seed cells in a 8-well Lab-Tak II chambered coverglass at 5x104 ...

<ol>
	<li>Hamaguchi, K., Gaskins, H. R., Leiter, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIT-1,+a+pancreatic+beta-cell+line+established+from+a+transgenic+NOD/Lt+mouse.">NIT-1, a pancreatic beta-cell line established from a transgenic NOD/Lt mouse.</a> Diabetes. 40, 842-849 (1991).</li><li>Chen, J., Gusdon, A. M., Piganelli, J., Leiter, E. H., Mathews, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mt-Nd2a+modifies+resistance+against+autoimmune+Type+1+diabetes+in+NOD+mice+at+the+level+of+the+pancreatic+beta+cell.">mt-Nd2a modifies resistance against autoimmune Type 1 diabetes in NOD mice at the level of the pancreatic beta cell.</a> Diabetes. , (2010).</li><li>Scaduto, R. C., Grotyohann, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+mitochondrial+membrane+potential+using+fluorescent+rhodamine+derivatives.">Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives.</a> Biophys J. 76, 469-477 (1999).</li><li>Lemasters, J. J., Ramshesh, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+mitochondrial+polarization+and+depolarization+with+cationic+fluorophores.">Imaging of mitochondrial polarization and depolarization with cationic fluorophores.</a> Methods Cell Biol. 80, 283-295 (2007).</li><li>Abel, M., Krokowski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+immune-mediated+(type+1)+diabetes+mellitus:+potential+for+immunotherapy.">Pathophysiology of immune-mediated (type 1) diabetes mellitus: potential for immunotherapy.</a> BioDrugs. 15, 291-301 (2001).</li><li>Mathews, C. E., Graser, R. T., Savinov, A., Serreze, D. V., Leiter, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unusual+resistance+of+ALR/Lt+mouse+beta+cells+to+autoimmune+destruction:+role+for+beta+cell-expressed+resistance+determinants.">Unusual resistance of ALR/Lt mouse beta cells to autoimmune destruction: role for beta cell-expressed resistance determinants.</a> Proc Natl Acad Sci U S A. , 235-240 (2001).</li><li>Szabadkai, G., Duchen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+mediated+cell+death+in+diabetes.">Mitochondria mediated cell death in diabetes.</a> Apoptosis. 14, 1405-1423 (2009).</li><li>Ly, J. D., Grubb, D. R., Lawen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial+membrane+potential+(deltapsi(m))+in+apoptosis;+an+update.">The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update.</a> Apoptosis. 8, 115-128 (2003).</li><li>Dudek, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+T-cells+from+T-cell+receptor+transgenic+NOD8.3+mice+destroy+beta-cells+via+the+perforin+and+Fas+pathways.">Cytotoxic T-cells from T-cell receptor transgenic NOD8.3 mice destroy beta-cells via the perforin and Fas pathways.</a> Diabetes. 55, 2412-2418 (2006).</li><li>Pakala, S. V., Chivetta, M., Kelly, C. B., Katz, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+autoimmune+diabetes+the+transition+from+benign+to+pernicious+insulitis+requires+an+islet+cell+response+to+tumor+necrosis+factor+alpha.">In autoimmune diabetes the transition from benign to pernicious insulitis requires an islet cell response to tumor necrosis factor alpha.</a> J Exp Med. 189, 1053-1062 (1999).</li></ol>

Experiments on animals were performed in accordance with the guidelines and regulations set forth by University of Florida Institutional Animal Care and Use Committee.

Cytotoxic T cell mediated beta cell death is the main pathophysiology of T1D 5. CML assays using 51Cr release allow us to study the degree of effector-target response 6. However, the detailed process and pathways of T cell-mediated beta cell death is still not fully understood. Since mitochondria are critical for beta cell function and death7, we focused on mitochondrial changes during the visual CML. Mitochondrial membrane potential dissipation is an early and irreversible...

<table border="1">
	<tbody>
 	<tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 	<td>Cr-51</td>
 <td>Perkin Elmer</td>
 <td>NEZ030500IMC</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>PBS</td>
 <td>Cellgro</td>
 <td>21-040-60</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Tetramethyl Rhodamine Methyl Ester (TMRM)</td>
 <td>Invitrogen</td>
 <td>T668</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Picogreen</td>
 <td>Invitrogen</td>
 <td>P7581</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>AI4 mimotope</td>
 <td>mimotopes.com</td>
 <td>amino acid sequence: YFIENYLEL</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>IL-2</td>
 <td>R &amp; D</td>
 <td>485-MI</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>DMEM</td>
 <td>Invitrogen</td>
 <td>11885</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>FBS</td>
 <td>HyClone</td>
 <td>SH30071.03</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Bovine Serum Albumin</td>
 <td>Sigma</td>
 <td>A8412</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>HEPES</td>
 <td>Cellgro</td>
 <td>25-060-Cl</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>MEM Non-Essential Amino Acids</td>
 <td>GIBCO</td>
 <td>11140</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Penicillin/Streptomycin</td>
 <td>Gemini</td>
 <td>400-109</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Phenol red-free DMEM</td>
 <td>Sigma</td>
 <td>D5303</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>CO2 Incubator</td>
 <td>Thermo Fisher</td>
 <td>HeraCell 150i</td>
 <td>Kept sterile for cell culture</td>
 </tr>
 <tr>
 	<td>Biological safety cabinet</td>
 <td>Thermo Fisher</td>
 <td>Forma 1440</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Disposable culture tubes, 6x50 mm Lime Glass</td>
 <td>Fisher</td>
 <td>14-958-A</td>
 <td>&nbsp;</td>
 </tr> 
 <tr>
 	<td>Gamma Counter</td>
 <td>Perkin Elmer</td>
 <td>Wizard 1470 Automatic Gamma Counter</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Confocal microscope </td>
 <td>Zeiss</td>
 <td>LSM 510 Meta Confocal</td>
 <td>With motorized stage and live cell chamber</td>
 </tr>
 <tr>
 	<td>Pipette (1ml, 200&mu;l, 20&mu;l,10&mu;l, 2&mu;l)</td>
 <td>Eppendorf</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Tubes (Amber)</td>
 <td>Fisher</td>
 <td>50819772</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Centrifuge</td>
 <td>Sorvall Legend RT+</td>
 <td>75004377</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Hemacytometer</td>
 <td>Fisher Scientific</td>
 <td>267110</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Upright Microscope</td>
 <td>Zeiss</td>
 <td>Axioskop40</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>NOD.Cg-Rag1tm1Mom Tg(TcraAI4)1Dvs/DvsJ Mice</td>
 <td>The Jackson Laboratory</td>
 <td>004347</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>NOD.Cg-Rag1tm1Mom Tg(TcrbAI4)1Dvs/DvsJ Mice</td>
 <td>The Jackson Laboratory</td>
 <td>004348</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>NOD-AI4&alpha;/ &beta; F1 mice</td>
 <td>N/A</td>
 <td>N/A</td>
 <td>Bred in UF animal facility</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

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

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

methods-to-assess-beta-cell-death-mediated-by-cytotoxic-t-lymphocytes

Research

JoVE Journal

Immunology and Infection

13.3K ビュー.  University of Florida. 細胞媒介のlymphocytotoxicity（CML）アッセイは、in vitroにおける自己反応性応答と細胞死の研究のメカニズムをテストするために使用することができます。しかし、蛍光染料、型と細胞死だけでなく、利用経路の動態と生きた細胞の共焦点顕微鏡イメージング技術を用いてより詳細に調べることができる。