Name: elispot assay for single-cell immune detection
Uploaded: 2023-04-30T07:08:00.000Z
Duration: 11 min 53 s
Channel: JoVE

K12 Standards

Texas Essential Knowledge and Skills

High School

Earth Systems Science

112.49.c.12.

112.49.c.12.A.

elispot-assay-detection-of-ifn-secreting-splenocytes

ELISPOT Assay: Detection of IFN-&#947; Secreting Splenocytes

1. 准备工作
缓冲液与试剂
<ol>
	<li>无钙无镁的无菌磷酸盐缓冲液（PBS）</li>
	<li>包被缓冲液——无菌 PBS 或碳酸盐缓冲液</li>
	<li>检测稀释液——含10%胎牛血清（FBS）的PBS</li>
	<li>细胞培养基 - RPMI 1640，含10% FBS、青霉素/链霉素 &amp;#38; L-谷氨酰胺</li>
	<li>洗涤缓冲液——含0.05% Tween20的PBS</li>
	<li>双蒸水（ddH₂O）2O</li>
	<li>检测底物——100 mg AEC（3-氨基-9-乙基-咔唑）溶于10 mL DMF（N,N-二甲基甲酰胺）中。</li>
</ol>
仪器设备
<ol start="8">
	<li>层流罩</li>
	<li>加湿培养箱（设定为 37°C，5% CO2）</li>
	<li>自动 ELISPOT 读板仪或解剖显微镜</li>
</ol>
材料
<ol start="11">
	<li>ELISPOT 板</li>
	<li>无菌和非无菌储液槽</li>
	<li>移液器及吸头</li>
	<li>无菌刻度移液管</li>
	<li>无菌锥形聚丙烯管</li>
	<li>两个用于洗板的挤压瓶</li>
</ol>
特定检测用试剂
<ol start="17">
	<li>细胞——原代细胞或细胞系（此处使用C57BL/6小鼠的脾细胞）</li>
	<li>刺激物——有丝分裂原或抗原（此处使用佛波酯12-肉豆蔻酸13-乙酸酯（PMA，50 ng/mL）和离子霉素（1 µM））</li>
	<li>一抗——生物素标记的抗细胞因子检测抗体（用检测稀释液稀释至2 µg/mL）</li>
	<li>二抗——链霉亲和素-辣根过氧化物酶（SAv-HRP）</li>
</ol>
2. 实验步骤
包被
<ol>
	<li>在无菌条件下并在超净工作台内，将纯化的抗细胞因子捕获抗体用无菌包被缓冲液稀释至终浓度为 0.5–4.0 µg/mL。（注意：IFN-γ 和 IL-6 使用 5 µg/mL。）</li>
	<li>将捕获抗体溶液以 100 µL/孔转移至 ELISPOT 板中。</li>
	<li>用板盖覆盖板子并密封，防止蒸发。</li>
	<li>在 4°C 下孵育过夜。</li>
</ol>
封闭
<ol start="5">
	<li>次日，在超净台中取出ELISPOT板。迅速将板倒置在无菌擦拭纸上，以去除每孔中的捕获抗体溶液。</li>
	<li>然后，向每孔加入200 µL细胞培养基。此步骤可阻断检测过程中非特异性结合。</li>
	<li>盖上板盖，将板在37°C下孵育2小时。</li>
</ol>
细胞铺板与活化
<ol start="8">
	<li>在平板孵育的同时，用细胞培养基配制含有 50 ng/mL PMA 和 1 µM 离子霉素的 2X 有丝分裂原溶液。</li>
	<li>然后，将靶细胞悬液调整至储存浓度为 2 × 106 个细胞/mL。</li>
	<li>孵育结束后，在超净工作台内将平板迅速倒置在无菌擦拭纸上，移除各孔中的细胞培养基。</li>
	<li>接下来，对储存的细胞悬液进行 2 倍系列稀释。首先，将 200 µL 已配制的细胞悬液原液加入 ELISPOT 板最上一排的各孔中。</li>
	<li>然后，在含有细胞原液的下一排开始，向接下来的五排每孔加入 100 µL 新鲜细胞培养基。</li>
	<li>随后，通过移液器将 100 µL 细胞悬液从最上一排转移至其正下方的一排，进行 2 倍系列稀释。为确保细胞均匀分布，需轻柔地上下吹打溶液以充分混匀。</li>
	<li>对剩余的四排重复此操作。</li>
	<li>第六排仅保留培养基，作为空白对照组。</li>
	<li>接着，向板中前五排的实验孔加入 100 µL 已配制的有丝分裂原溶液；在对照孔及第六排中，则加入 100 µL 不含有丝分裂原的细胞培养基。</li>
	<li>盖好板盖，将平板置于 37°C、5% CO2 的培养箱中孵育 20–48 小时。（注意：检测 IL-2 和 TNF-α 通常 20–24 小时已足够，而检测 IL-4 和 IFN-γ 时，48 小时为最佳孵育时间）。</li>
</ol>
检测
一抗
<ol start="18">
	<li>用检测稀释液将生物素标记的抗细胞因子检测抗体稀释至浓度为 2 µg/mL。</li>
	<li>此时配制 20–25 mL 洗涤缓冲液，即在 PBS 中加入 0.05% Tween-20 并混匀。</li>
	<li>孵育完成后，打开板盖，迅速将板倒置于水槽上方，以彻底移除各孔中的液体。（注意：此后不再需要保持板的无菌状态）。</li>
	<li>随后，向每孔加入约 200 µL 洗涤缓冲液进行洗涤。通过迅速倒置并在水槽上方轻弹板子，排出液体。重复此步骤，共洗涤五次。</li>
	<li>接着，向每孔加入 100 µL 稀释后的生物素标记抗细胞因子检测抗体溶液。于室温孵育 2 小时，或在 4°C 过夜孵育。</li>
</ol>
二抗
<ol start="23">
	<li>孵育完成后，将板倒置并在水槽上方轻弹，排出检测抗体。</li>
	<li>同前，用约 200 µL 洗涤缓冲液洗涤板 5 次，每次洗涤之间彻底排出液体。</li>
	<li>随后向每个孔中加入 100 µL 稀释后的链霉亲和素-辣根过氧化物酶溶液（用检测稀释液稀释至预先确定的最佳浓度）。</li>
	<li>盖好板盖，在 37°C 下室温孵育 1.5–2 小时。</li>
</ol>
底物
<ol start="27">
	<li>孵育结束后，在使用前不超过15分钟，首先根据制造商说明书激活AEC底物溶液。</li>
	<li>接着，弃去各孔中的内容物，并用洗涤缓冲液洗涤平板5次，方法同前。</li>
	<li>然后，立即向每孔中加入100 µL配制好的AEC底物溶液。</li>
	<li>在室温下孵育平板约10–20分钟，同时监测斑点的显色情况。</li>
	<li>通过用水冲洗平板并在水槽上方轻弹平板以终止反应。</li>
	<li>将平板在纸巾上轻轻吸干，并置于空气中过夜晾干，或直至完全干燥。移除平板下方的塑料托盘有助于加快干燥过程。</li>
</ol>
3. 数据采集与分析
<ol>
	<li>干燥后，斑点即可使用自动化酶标仪进行计数。此处使用的是 CTL Immunospot 酶标仪，但本方案可适用于任何型号的酶标仪。</li>
	<li>首先打开仪器，然后开启计算机。接着，打开 CTL 程序并点击 &#34;扫描次数&#34;</li>
	<li>推 &#34;弹出&#34; 使托盘从机器中伸出。然后，取下塑料适配器，并对齐排 &#34;A&#34; 在ELISPOT板和适配器上</li>
	<li>选择文件的保存名称和位置，并将酶标板加载到托盘上。</li>
	<li>然后，单击 &#34;加载&#34; 在软件上操作并关闭机器侧面的门。</li>
	<li>按压 &#34;计数后开始。&#34; 确保文件已保存，然后打开质量控制 &#34;质量控制&#34; 用于分析数据并计数斑点数量的软件。</li>
</ol>
注意事项：
<ol type="a">
	<li>细胞的最少数量应在预实验中确定。每个孔的最佳斑点数量约为50个。如果加入的细胞过多，则难以分辨清晰的斑点。此外，细胞可能发生重叠，无法在膜上形成单层，从而降低检测水平。</li>
	<li>在优化实验时，应考虑目标蛋白的预期表达水平。表达水平越低，每孔所需的细胞数量就越多。</li>
	<li>与ELISA不同，建议手动洗涤板孔，而不是使用洗板机。ELISPOT板较为脆弱，应避免刺穿PVDF膜。</li>
	<li>在孵育期间应尽量减少板的移动，以免造成斑点模糊。</li>
	<li>板应避光保存，因为直接暴露在光线下会导致斑点褪色。</li>
</ol>

ELISPOT 检测法可通过测定分泌特定分析物的细胞数量来评估免疫细胞的活化状态。斑点的大小和强度可反映每个细胞产生分析物的量。上述方案详细描述了单一种细胞因子的检测。然而，近年来的技术进展已显著提升了该检测方法的应用范围。目前，可通过使用荧光检测试剂，在同一孔中同时检测多种分析物，从而实现对分泌一种或两种分析物的不同细胞亚群的识别与检测。

elispot实验：检测分泌ifn-γ的脾细胞

来源：Tonya J. Webb1 
1 马里兰大学医学院微生物学与免疫学系及Marlene和Stewart Greenebaum综合癌症中心，美国马里兰州巴尔的摩市 21201
ELISPOT 是一种标准化且可重复的检测方法，用于检测细胞免疫应答。该检测利用基于酶联免疫吸附试验（ELISA）的方法来检测单个细胞的免疫反应，这些反应可通过斑点可视化，因此得名 ELISPOT。ELISPOT 于 1983 年由 Czerkinsky 首次描述，作为一种计数产生抗原特异性免疫球蛋白的 B 细胞杂交瘤数量的方法（1）。同一研究团队进一步开发了该检测方法，用于测量产生细胞因子的 T 淋巴细胞的频率。如今，ELISPOT 已成为临床试验和疫苗候选物中评估抗原特异性 T 细胞免疫的金标准。例如，在接种疫苗后或感染期间，浆细胞和记忆 B 细胞会分泌提供保护作用的抗体。通常，这些 B 细胞反应通过测定血清中抗原特异性抗体的滴度来评估。然而，这种分析方法（通常通过 ELISA 测定）可能无法涵盖记忆 B 细胞，而这些细胞即使在血清抗体水平不可检测的情况下也可能存在。此外，已有充分证据表明，循环中的记忆 B 细胞对于病原体再次暴露后所观察到的快速且具有保护性的抗体反应至关重要，因此能够检测这些细胞十分关键。因此，为了明确评估抗原特异性的记忆 B 细胞反应，应同时使用 ELISA 和 ELISPOT 方法（2）。
ELISPOT 实验采用含有膜包被孔的板，孔壁上包被有抗体，用于捕获目标分泌蛋白。随后，将细胞和刺激物加入板中以诱导蛋白产生。分泌的蛋白被包被在表面的抗体所捕获。经过适当的孵育时间后，去除细胞，再使用一种针对不同表位（相对于捕获抗体）的生物素标记抗体来检测被固定的分泌分子。接着加入链霉亲和素过氧化物酶，然后加入底物，从而实现对斑点的检测（图1）。该实验的优势在于能够定量产生目标蛋白的细胞数量。重要的是，可以评估产生特定蛋白的细胞总数是否发生变化，或细胞群体中的单个细胞是否产生更多的蛋白。此外，该方法可提供有关动力学的信息，并可用于评估整体免疫激活（有丝分裂原刺激）与抗原特异性反应（抗原刺激）之间的关系。在有丝分裂原或抗原特异性激活后，ELISPOT 实验能够从30万个细胞中检测出一个活化的细胞。
<img alt="ELISpot检测示意图；抗体包被、细胞加入、检测及斑点计数的过程。" class="xfigimg" src="/files/ftp_upload/10497/10497fig1.jpg" data-altupdatedat="1761830439000" data-alt="图1"> 
图1：ELISPOT实验流程概览。
该检测方法的主要优势在于：a. 简便性——该方案相对简单直接，无需专业技术经验；b. 灵敏度——可在单细胞水平检测免疫细胞，且所需细胞数量远少于流式细胞术等其他方法；c. 功能性——可提供有关免疫功能的定量数据。
本实验演示了用于检测分泌 IFN-γ 的脾细胞的 ELISPOT 方案，但如上所述，该检测也可用于评估 B 细胞的抗体分泌（3）。

在本ELISPOT实验中，分析了来自野生型小鼠和荷瘤小鼠的脾白细胞中IFN-γ的表达。图2A展示了实验结果的可视化图像。绿色数字表示每孔中的斑点数量（TNTC表示“多到无法计数”）。请注意，随着细胞浓度降低，斑点数量减少。
<img alt="ELISpot 检测结果，免疫学实验，显示细胞因子检测孔中形成的斑点。" src="/files/ftp_upload/10497/10497fig2a.jpg" data-altupdatedat="1761830439000" data-alt="图 2A"> 
图 2A：荷瘤小鼠中免疫应答降低。 <a href="https://www.jove.com/files/ftp_upload/10497/10497fig2alarge.jpg" target="_blank">请点击此处查看该图的放大版本。</a>
<p class="jove_content" ...

ELISPOT实验：检测分泌IFN-γ的脾细胞

来源：Tonya J. Webb1  
1 马里兰大学医学院微生物学与免疫学系，Marlene and Stewart Greenebaum ...

酶联免疫斑点（ELISPOT）检测是一种用于分析机体对病原体或细胞损伤免疫应答的方法。该方法通过检测免疫细胞分泌的特定蛋白质，实现对不同类型免疫细胞活化状态的定量分析。例如，ELISPOT 常用于检测T细胞在接触外来抗原后分泌的细胞因子，从而评估T细胞的免疫应答反应。对于基于细胞因子的ELISPOT实验，首先用针对目标细胞因子的捕获抗体包被ELISPOT微孔板。抗体包被完成后，将T细胞加入微孔中，并通过外部刺激物（例如抗CD3抗体）进行刺激。随后，细胞分泌目标细胞因子，该因子会立即被包被在板上的捕获抗体所固定。由于蛋白质在活细胞分泌后即刻被捕获，未经历稀释或降解，因此该检测方法具有很高的准确性。在目标细胞因子被固定后，加入检测抗体，该抗体也会结合已捕获的细胞因子。ELISPOT 技术还可通过分析记忆B细胞产生特异性抗体的情况，来定量感染或疫苗接种后的记忆B细胞。在基于抗体的 ELISPOT 中，使用特定抗原替代抗体，用于捕获步骤（此时抗原被结合到板上）或检测步骤（此时抗原用于在捕获后识别目标抗体）。在所有该技术的变体中，无论是用于T细胞还是B细胞，检测用抗体或抗原均被生物素化，从而能够与链霉亲和素偶联的检测酶（如辣根过氧化物酶）结合。随后，加入过氧化物酶的底物 AEC 后，会产生深色、不溶性的沉淀物。该沉淀物标记了被捕获蛋白的位置，每个分泌细胞形成一个可见的斑点，可通过 ELISPOT 读板仪或显微镜进行定量。斑点的大小可相对反映每个细胞分泌蛋白的量。该检测方法能够从单个细胞水平检测免疫应答，即使在相对较少的分泌细胞亚群中也具有灵敏性，因此适用于在细胞水平研究免疫应答。在本视频中，您将学习如何进行ELISPOT实验，并对代表分泌细胞的斑点进行定量分析。整个实验过程中，应在超净工作台内操作并佩戴手套，以确保无菌条件。本方案中的所有计算均基于一个96孔板所需的体积。首先，稀释抗细胞因子捕获抗体。为此，将10毫升缓冲液转移至无菌的15毫升锥形管中。然后，使用移液器向缓冲液中加入10微升浓度为1毫克/毫升的单克隆抗体，制成终浓度为1微克/毫升的溶液。接着，将捕获抗体溶液倒入无菌储液槽中，并使用多通道移液器将100微升溶液分配至96孔ELISPOT板的每孔中。用酶标板盖覆盖酶标板，密封以防止蒸发，并在4°C下孵育过夜。次日，在超净工作台内打开ELISPOT板。迅速将酶标板倒置在无菌擦拭纸上，以去除各孔中的捕获抗体溶液。接着，使用多通道移液器向每孔加入200 µL细胞培养基。此步骤可阻断检测过程中的非特异性结合。重新盖上酶标板盖，于37°C培养箱中孵育2小时。在培养板孵育的同时，通过将1微升PMA和20微升离子霉素加入10毫升细胞培养基中，配制2倍浓度的有丝分裂原溶液，使PMA的终浓度为15纳克/毫升，离子霉素的终浓度为1微摩尔。此时也应在无菌操作台中制备小鼠脾细胞的细胞悬液。使用显微镜和血细胞计数板测定细胞浓度，并调整总体积，直至达到每毫升两百万个细胞的储备浓度。孵育完成后，迅速将培养板倒置在无菌擦拭纸上，以去除每孔中的细胞培养基。接着，向ELISPOT板最上方一行的各孔中加入200 µL制备好的细胞悬液储备液。实验需设置三个重复孔，使得每种检测的细胞类型均接种于一组相邻的三列孔中。在此下方的五排孔中，每孔加入100 µL普通细胞培养基，这些孔位于含有细胞储备液的孔排之下。接下来，通过移液将顶部一行的细胞悬液100微升加入正下方的一行中，轻轻吹打溶液上下混合，使细胞均匀分布。对剩余各行重复此操作，每一步都将前一行的100微升转移至下一行，直至完成第五行的连续稀释。第六行仅保留细胞培养基，作为空白对照。为了刺激板中实验孔内的细胞，向第一至第五行各孔中的细胞悬液加入100微升已配制好的有丝分裂原溶液。务必保持作为对照的第六行不接受刺激。盖上盖子，将培养板置于37°C、5% CO2条件下孵育24至48小时。配制稀释的生物素化抗细胞因子检测抗体。首先，将5毫升10%胎牛血清加入45毫升磷酸盐缓冲液（PBS）中，制备50毫升检测稀释液。接着，用检测稀释液将检测抗体稀释至浓度为2微克/毫升。同时，配制20至25毫升洗涤缓冲液，即在PBS中加入0.05% Tween-20并混匀。孵育完成后，打开酶标板盖，迅速倒置酶标板以彻底移除各孔中的液体。通过向每个孔中加入约 200 µL 洗涤缓冲液来洗涤酶标板。将酶标板在水槽上方迅速倒置并轻弹，以甩出孔内液体。重复此步骤四次，共完成五次洗涤。接下来，向每个孔中加入 100 µL 稀释后的检测抗体溶液，盖上盖子，在室温下孵育两小时。孵育结束后，将酶标板在水槽上方倒置并轻弹，甩出孔中的检测抗体溶液。同前，用洗涤缓冲液洗涤平板五次，每次洗涤之间彻底甩净液体。最后一次洗涤后，根据生产商说明书稀释链霉亲和素-辣根过氧化物酶溶液，配制工作液。随后，在平板各孔为空的状态下，向每孔加入100 µL稀释后的链霉亲和素-辣根过氧化物酶溶液。盖上盖板，于室温孵育两小时。孵育后，在使用前不超过15分钟时，激活预先配制的AEC底物溶液。弃去各孔中的内容物，并用洗涤缓冲液按前述方法洗涤5次。随后，立即向每孔中加入100 µL已配制好的AEC底物溶液。将酶标板置于室温下显色约10至20分钟，并同时监测斑点的形成情况。这些斑点将呈现为孔表面的小型深色圆圈。显色完成后，用清水冲洗酶标板并在水槽上方轻轻弹去多余液体以终止反应。将酶标板在纸巾上轻压吸干，并置于空气中过夜或直至完全干燥。移除酶标板底部的塑料托盘有助于加快干燥过程。干燥后，即可使用自动酶标仪对斑点进行计数。此处使用 CTL ImmunoSpot 读板仪，但本方案可适用于任何读板仪。然后，打开 CTL 程序并点击“Scan Count”。按下“Eject”按钮，使托盘从仪器中伸出。取下塑料适配器，并将 ELISPOT 板和适配器上的 A 行对齐。为待保存的文件选择文件名和存储位置，然后将板子和适配器放入托盘中。在软件中点击“Load”，并关闭仪器侧面的门。随后，点击“Start After Counting”。确保文件已保存后，打开质控（QC）软件以分析数据并统计斑点数量。将此数据导出为 Excel 文件。分析完成后，点击“Eject”以取出板子。在本实验中，将野生型小鼠和荷瘤小鼠的细胞接种并分析其干扰素γ（IFN gamma）。请注意，随着细胞浓度的降低，斑点数量也随之减少。通常，ELISPOT 数据以每接种细胞数量对应的斑点数来表示。在本示例中，斑点数量以柱状图显示，各个相应的细胞浓度列在横轴上。请注意，斑点数量表示特定细胞群体中活化细胞占总细胞数的比例。

Education

教育

JoVE Science Education

Advanced Biology

高级生物学

Immunology

免疫学

ELISPOT 分析:检测 IFN-- 分泌性细胞

elispot assay for single-cell immune detection

ELISPOT is a sensitive assay for detecting immune cells that release a specific protein. It is based on an ELISA, or enzyme-linked immunosorbent assay, but it shows single-cell responses as visible spots. The method was first described in 1983 by Czerkinsky to count B cell hybridomas that produce antigen-specific immunoglobulins.

The assay was later adapted to measure cytokine-producing T lymphocytes. Because of that, ELISPOT is now a gold standard for measuring antigen-specific T cell immunity in clinical trials and vaccine candidates. It can also help study B cell responses after vaccination or infection, including plasma cells and memory B cells that secrete protective antibodies.

B cell responses are often measured with serum antibody titers by ELISA. However, that approach may miss memory B cells, especially when serum antibody levels are not detectable. Since circulating memory B cells are important for a rapid and protective response after re-exposure to a pathogen, both ELISA and ELISPOT can be needed to assess antigen-specific memory B-cell responses.

The assay uses a plate with membrane-lined wells coated with capture antibodies. Cells and a stimulus are added to trigger protein production. The secreted proteins bind to the antibodies on the surface. After incubation, the cells are removed, and a second biotinylated antibody binds a different epitope on the secreted molecule. Streptavidin peroxidase and a substrate are then added so the spots can be seen.

ELISPOT is useful because it can count the number of cells making the protein of interest. It can also show whether more cells are producing a protein or whether individual cells are producing more of it. The assay can give information about kinetics and can compare overall immune activation from mitogen stimulation with antigen-specific responses. It can detect one activated cell among 300,000 cells after either type of activation.

The main advantages of ELISPOT are simplicity, sensitivity, and functionality. The protocol is relatively straightforward and does not require advanced technical expertise. It detects immune cells at the single-cell level and uses very few cells compared with methods such as flow cytometry. It also provides quantitative data about immune function. This lab exercise focuses on detecting IFN-γ-secreting splenocytes, but the same assay can also be used to measure antibody secretion by B cells.

ELISPOT Assay for Single-Cell Immune Detection

elispot-assay-for-single-cell-immune-detection

flow cytometry and fluorescence-activated cell sorting (facs): isolation of splenic b lymphocytes

flow-cytometry-fluorescence-activated-cell-sorting-facs-isolation

Sorting B Cells from Spleen with FACS

Flow cytometry and FACS can be used to separate B lymphocytes from a mixed spleen cell sample. The immune system protects the body from infectious organisms and other invaders. White blood cells, also called leukocytes, are the main cells involved in this defense. Leukocytes can be grouped into subpopulations such as myeloid cells, lymphocytes, and dendritic cells. These groups can be distinguished by biological, physical, and functional features. Cell size, granularity, and secretion are...

magnetic activated cell sorting (macs): isolation of thymic t lymphocytes

magnetic-activated-cell-sorting-macs-isolation-of-thymic-t-lymphocytes

Enriching Thymic T Cells with Magnetic Sorting

Thymic T cell enrichment with magnetic sorting helps isolate a targeted immune cell group from a mixed sample. The immune system relies on many cell types, and lymphocytes are a major part of adaptive immunity. B cells usually respond to extracellular infections, such as bacterial infections, while T cells usually respond to intracellular infections, such as viral infections. Magnetic sorting enriches selected cells by using magnetic beads and antibodies against cell surface proteins. First,...

elisa assays: indirect, sandwich, and competitive

elisa-assays-indirect-sandwich-and-competitive

ELISA Methods for Detecting Antigens

ELISA methods detect antigens, antibodies, proteins, hormones, peptides, and other biomolecules in biological samples. Enzyme-linked immunosorbent assay is highly sensitive because it can detect very low antigen levels. It works by using an antigen-antibody interaction, then an enzyme-linked antibody turns a colorless substrate into a colored or fluorescent signal that a plate reader can measure. Different ELISA formats are used for different sample types and antibody setups. Indirect ELISA is...

elispot assay: detection of ifn-&#947; secreting splenocytes

ELISPOT is a sensitive assay for detecting immune cells that release a specific protein. It is based on an ELISA, or enzyme-linked immunosorbent assay, but it shows single-cell responses as visible spots. The method was first described in 1983 by Czerkinsky to count B cell hybridomas that produce antigen-specific immunoglobulins. The assay was later adapted to measure cytokine-producing T lymphocytes. Because of that, ELISPOT is now a gold standard for measuring antigen-specific T cell...

immunohistochemistry and immunocytochemistry: tissue imaging via light microscopy

immunohistochemistry-immunocytochemistry-tissue-imaging-via-light

Antibody Staining of Tissue and Cells

Antibody staining of tissue and cells lets scientists see where a specific antigen is located. Immunohistochemistry, or IHC, uses thin tissue sections. Immunocytochemistry, or ICC, uses isolated cells or cells grown in culture. Both methods rely on antibodies that carry a chemical or fluorescent tag, such as peroxidase or rhodamine, so the target antigen can be detected under a light microscope. IHC keeps the structure of the whole tissue in place while the sample is stained. That makes it...

antibody generation: producing monoclonal antibodies using hybridomas

antibody-generation-producing-monoclonal-antibodies-using-hybridomas

Hybridoma Cell Culture for Monoclonal Antibodies

Hybridoma cell culture is used to produce monoclonal antibodies, or mAbs, with a known antigen specificity. Monoclonal antibodies come from a single clone of cells, so they recognize one target more precisely than polyclonal antibodies, which are a mixed group of antibodies against different antigenic determinants. That difference matters when similar molecules share epitopes, or antigen sites, that can confuse an antibody test. Polyclonal antisera can react with more than one related antigen.

immunofluorescence microscopy: immunofluorescence staining of paraffin-embedded tissue sections

immunofluorescence-microscopy-immunofluorescence-staining-paraffin

Fluorescent Antibody Staining of Tissue Slides

Fluorescent antibody staining lets scientists see specific proteins in thin tissue sections on glass slides. It is used to study normal tissue structure and to better understand how disease works. In many cases, the tissue comes from patient biopsies or from experimental in vivo models. The tissue is usually fixed in formalin or paraformaldehyde and embedded in paraffin wax before it is cut. This preservation method supports long-term storage and makes sectioning possible. A microtome is used...

confocal fluorescence microscopy: a technique to determine the localization of proteins in mouse fibroblasts

confocal-fluorescence-microscopy-technique-to-determine-localization

Confocal Microscopy for Protein Localization

Confocal fluorescence microscopy is used to show where proteins are located inside cells with better detail than wide-field epifluorescence microscopy. It improves image clarity by scanning the sample with lasers and using a pinhole to block out-of-focus light. This makes it easier to see whether a signal comes from the cell surface or from inside the cell. The method relies on fluorescence, which happens when a molecule absorbs light and then gives off a new photon. A fluorophore is a...

immunoprecipitation-based techniques: purification of endogenous proteins using agarose beads

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Protein Pull-Down with Antibodies and Beads

Immunoprecipitation uses antibodies and Protein A/G agarose beads to isolate a specific protein from a cell lysate. It is also called a pull-down assay. The term "immuno" refers to the antibody, and "precipitation" means pulling a substance out of solution. The target protein can be endogenous or recombinant. Recombinant proteins often carry epitope tags such as myc or flag, which makes purification easier because antibodies against these tags are usually strong and effective. Antibodies...

cell cycle analysis: assessing cd4 and cd8 t cell proliferation after stimulation using cfse staining and flow cytometry

cell-cycle-analysis-assessing-cd4-cd8-t-cell-proliferation-after

Tracking T Cell Division with CFSE and Flow Cytometry

Tracking T cell division with CFSE and flow cytometry shows how immune cells respond after stimulation. The cell cycle is a normal process of life. During it, a cell changes step by step and divides into two daughter cells. This happens throughout life when the body needs new cells for growth and repair. Cells that are not cycling are in the G0 phase, which means they are quiet. Once a cell enters the cycle, it moves through G1, S, G2, and M phases. G1 is a checkpoint for the resources needed...

adoptive cell transfer: introducing donor mouse splenocytes to a host mouse and assessing success via facs

adoptive-cell-transfer-introducing-donor-mouse-splenocytes-to-host

Tracking Transferred Mouse Splenocytes by FACS

Adoptive cell transfer is a method for moving cells into a host animal to study biology or help treat disease. Researchers can use it to follow cell movement and distribution, or to test how transferred cells behave in a mouse model. Tracking systems such as cell surface markers and CFSE staining can help show where the cells go after transfer. This approach has several uses in mouse studies. It can support cancer research by transferring specific cell populations as an experimental treatment...

assay for cell death: chromium release assay of cytotoxic ability

assay-for-cell-death-chromium-release-assay-of-cytotoxic-ability

Chromium Release Assay for Immune Cell Killing

The chromium release assay measures how well immune cells kill target cells in the lab. It is used to test the cytotoxic ability of T cells, NK cells, and other effector cells against infected cells or tumor cells. This assay works by labeling target cells with chromium-51, a radioactive tracer called Na2 51CrO4. The label quickly marks cellular proteins inside the target cells. When effector cells damage the target-cell membrane, the labeled proteins are released into the culture supernatant...

immunology

该系列涵盖了免疫学实验室的许多主要技术，包括免疫细胞的标签和分类。它还演示了免疫细胞和抗体的增殖方法，以及包括ELISA在内的免疫活性的常见测定方法。最后，演示了免疫组织和细胞样本的染色和成像。

Learn how ELISPOT detects single immune cells by spotting IFN-γ secretion in splenocytes, with uses in T cell and B cell response analysis.

ELISPOT Assay for Single-Cell Immune Detection | JoVE K12

Tonya J. Webb

Department of Microbiology and Immunology

University of Maryland School of Medicine and the Marlene and Stewart Greenebaum Comprehensive Cancer Center

Concepts

Coating and Blocking the Plate

Plating and Activating Cells

Detection

Substrate

Data Acquisition and Analysis

Results

Source: Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum ...

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ELISPOT Assay for Single-Cell Immune Detection

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