Name: visualizing antigen specific cd4+ t cells using mhc class ii tetramers
Uploaded: 2009-03-06T00:00:00
Description: Watch this Scientific Journal Video about Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers at JoVE.com

<p class="jove_content">我们感谢他们在拍摄和视频制作的关键作用迪克弗利和Leigh金博尔。 </p>

<p class="jove_title"> 1。外周血单个核细胞（PBMC）隔离</p><ol><li>获取血液样本 - 血液应收集在注射器或血管和（1:50的比例）与肝素的抗凝固，防止凝血。预计产量约1 × 10 <sup>6</sup>外周血单个核细胞每毫升血液-其中约40％将CD4阳性（CD4 +）T细胞。 </li><li>分装成50 ml锥形管，每管25毫升的血液。如果血液分离，轻轻混匀分发血浆前aliquoting </li><li>添加PBS中，使总体积至40毫升和11毫升的聚蔗糖底图，进入血液管插入，并仔?...

<ol>
	<li>Novak, E. J., Liu, A. W., Nepom, G. T., Kwok, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MHC+Class+II+tetramers+permit+detection+and+characterization+of+peptide-specific+human+CD4++T+cells+proliferating+to+influenza+A+antigen.">MHC Class II tetramers permit detection and characterization of peptide-specific human CD4+ T cells proliferating to influenza A antigen.</a> <em style='font-style: italic'>J. Clin. Invest</em>. <b>104</b>, R63-R67 (1999).</li><li>Novak, E. J., Liu, A. W., Gebe, J. A., Falk, B., Nepom, G. T., Falk, B., Koelle, D. M., Kwok, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetramer-guided+epitope+mapping:+rapid+identification+and+characterization+of+immunodominant+CD4++T+cell+epitopes+from+complex+antigens.">Tetramer-guided epitope mapping: rapid identification and characterization of immunodominant CD4+ T cell epitopes from complex antigens.</a> <em style='font-style: italic'>J. Immunol</em>. <b>166</b>, 6665-6670 (2001).</li></ol>

<p class="jove_content">了解免疫的CD4 + T细胞的作用是极其重要的。然而，抗原特异性CD4 + T细胞可以用传统方法难以发现和隔离。相比之下，MHC II类四聚体，使所需的抗原特异性CD4 + T细胞的直接可视化。这个视频演示的分离，纯化，并在体外扩增的CD4 + T细胞，并随后将其可视化，使用四聚体。 II类四聚四是荧光蛋白偶联物的水溶性生物素化的II类的α/围绕一个荧光标记的链霉亲和的核心（图4）结合β二聚体组成。在本?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Class 2 Laminar flow biosafety cabinet</td>
<td>equipment</td>
<td>Thermo Forma</td>
<td>1200</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>50 mL conical Tube</td>
<td>equipment</td>
<td>VWR</td>
<td>47747-182</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>1X PBS (Ca/Mg free)</td>
<td>Reagent</td>
<td>HyClone</td>
<td>SH30028.02</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>Ficoll-paque PLUS</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1440-03</td>
<td>foil wrapped to protect from light</td>
</tr>
<tr>
<td>Pipet Aid XP</td>
<td>equipment</td>
<td>Drummond</td>
<td>4-000-101</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>10 mL pipet</td>
<td>equipment</td>
<td>Corning/Costar</td>
<td>CLS4492</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>Aerosolve Canisters</td>
<td>equipment</td>
<td>Beckman</td>
<td>359481</td>
<td>with 50 mL inserts</td>
</tr>
<tr>
<td>Centrifuge</td>
<td>equipment</td>
<td>Beckman</td>
<td>GS-6R</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>Transfer pipets </td>
<td>equipment</td>
<td>Samco</td>
<td>202-20S</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>Pasteur pipets</td>
<td>equipment</td>
<td>VWR</td>
<td>14672-200</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>Hemolytic Buffer</td>
<td>Reagent</td>
<td>N/A</td>
<td>N/A</td>
<td>8.3 g/L NH4Cl, 1.0 g/L NaHCO3, 0.04 g/L disodium EDTA</td>
</tr>
<tr>
<td>Trypan blue</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T6146</td>
<td>0.2% in PBS</td>
</tr>
<tr>
<td>Hemocytometer</td>
<td>equipment</td>
<td>VWR </td>
<td>15170-208</td>
<td>&nbsp;</td>
<tr>
<td>15 mL conical tube</td>
<td>equipment</td>
<td>VWR</td>
<td>05-527-90</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>Running Buffer</td>
<td>Reagent</td>
<td>N/A</td>
<td>N/A</td>
<td>PBS + 2mM EDTA + 5g/L BSA</td>
</tr>
<tr>
<td>MACS CD4+ T Cell Isolation Kit II</td>
<td>Reagent</td>
<td>Miltenyi Biotec</td>
<td>130-091-155</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>EasySep&reg;   Magnet</td>
<td>equipment</td>
<td>Stem Cell Technologies</td>
<td>18000</td>
<td>		</td>
</tr>
<tr>
<td>5 mL polypropylene tube</td>
<td>equipment</td>
<td>Falcon</td>
<td>352063</td>
<td>		</td>
</tr>
<tr>
<td>RPMI 1640</td>
<td>Reagent</td>
<td> Invitrogen</td>
<td>22400-089</td>
<td>with 25 mM HEPES</td>
</tr>
<tr>
<td>Pooled human serum</td>
<td>Reagent</td>
<td>N/A</td>
<td>N/A</td>
<td>drawn from healthy donors, heat inactivated and filtered</td>
</tr>
<tr>
<td>Pen Strep</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>1570-063</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>500mL 0.22 micron bottle top filter</td>
<td>equipment</td>
<td>Nalgene</td>
<td>595-3320</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>48-well plate</td>
<td>equipment</td>
<td>Costar</td>
<td>3548</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>37&deg;C CO<sub>2</sub> incubator</td>
<td>equipment</td>
<td>Sanyo Scientific</td>
<td>MCO-18AIC</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>20 mg/mL synthetic peptide</td>
<td>Reagent</td>
<td>N/A</td>
<td>N/A</td>
<td>Custom synthesis from any peptide vendor</td>
</tr>
<tr>
<td>5 mL FACS tube</td>
<td>equipment</td>
<td>Falcon</td>
<td>352008</td>
<td>Polystyrene</td>
</tr>
<tr>
<td>Peptide loaded class II tetramer (as described)</td>
<td>Reagent</td>
<td>N/A</td>
<td>N/A</td>
<td>Prepared by the BRI tetramer core</td>
</tr>
<tr>
<td>Anti-CD3</td>
<td>Reagent</td>
<td>BD pharmingen</td>
<td>347344</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>Anti-CD4</td>
<td>Reagent</td>
<td>eBioscience</td>
<td>17-0049-73</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>Anti-CD25</td>
<td>Reagent</td>
<td>eBioscience</td>
<td>11-0259-73</td>
<td>Or equivalent</td>
</tr>
<tr>
<td>Facs Calibur Flow Cytometer</td>
<td>equipment</td>
<td>BD Biosciences</td>
<td>342975</td>
<td>Or equivalent</td>
</tr>		</tbody>
		</table>
		</div>
	

visualizing antigen specific cd4+ t cells using mhc class ii tetramers

<p class="jove_content">主要组织相容性复合体（MHC）Ⅱ类四聚体可以直接可视化的抗原特异性CD4 + T细胞流式细胞仪。此方法依赖于非常具体的肽载入MHC和相应的T细胞受体之间的互动。虽然单一的MHC /肽分子的亲和力低，交叉连接链霉亲和的MHC /肽复合物增加亲和力的互动，使染色试剂的使用。由于CD4 + T细胞（〜1 30万为一个单一的特异性）的频率相对较低，本试验利用体外扩增步骤，以提高其检测阈值。纯化外周血单个核细胞经Ficoll底图。然后由消极的选择，使用生物素抗体鸡尾酒和抗生物素标记的磁珠分离CD4 +细胞。使用贴壁细胞的CD4细胞的一小部分，作为抗原提呈细胞，CD4 + T细胞在媒体扩大，加入的抗原肽和IL - 2。孵化一小时，随后在37℃染色使用表面抗体，如抗CD4，抗CD3和抗CD25扩大细胞与相应的Ⅱ类四聚体染色。标签后，细胞可以直接通过流式细胞仪分析。扩大CD4 +细胞中的四聚体阳性细胞通常形成一个独特的人口。四聚体阳性细胞通常CD25 +调节性和经常的CD4高。由于背景的四聚体染色水平可以有所不同，应该始终是积极的染色结果相比，相同的细胞与一个不相关的四聚体染色。这一基本检测的多种变化是可能的。四聚体阳性细胞可能为进一步的表型分析，列入酶联免疫斑点或增殖实验，或其他二次检测进行排序。一些团体也展示了合作，使用四聚体的抗细胞因子或抗FOXP3抗体染色。</p>

Watch this Scientific Journal Video about Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers at JoVE.com

Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

<p class="jove_content">此过程演示的纯化和体外扩增抗原特异性CD4 +从整体外周血T细胞及其可视化使用MHC II类四聚体。四聚体，允许直接与单个抗原特异性T细胞和定义MHC II类限制性的可视化。</p>

可视化抗原特异性CD4 + T细胞的MHC II类四聚四

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method ...

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