Name: tyramide signal amplification for the immunofluorescent staining of zbp1-dependent phosphorylation of ripk3 and mlkl after hsv-1 infection in human cells
Uploaded: 2022-10-20T14:00:00
Description: Watch this Scientific Journal Video about Tyramide Signal Amplification for the Immunofluorescent Staining of ZBP1-Dependent Phosphorylation of RIPK3 and MLKL After HSV-1 Infection in Human Cells at J

我们要感谢VIB生物成像核心的培训，支持和进入仪器园区。J.N.得到了法兰德斯研究基金会（FWO）的博士奖学金支持。J.M.小组的研究得到了奥德修斯II资助（G0H8618N），EOS INFLADIS（40007512），法兰德斯研究基金会（FWO）的初级研究资助（G031022N），CRIG青年研究者概念验证资助和根特大学的支持。P.V.组的研究得到了EOS MODEL-IDI（30826052），EOS INFLADIS（40007512），FWO高级研究资助（G.0C76.18N，G.0B71.18N，G.0B96.20N，G.0A9322N），Methusalem（BOF16 / MET_V / 007），iBOF20 / IBF / 039 ATLANTIS，抗癌基金会（F / 2016 / 865，F / 2020 / 1505），CRIG和GIGG联盟以及VIB的支持。

1. 生物素化酪胺的制备
<ol><li>从生物素-酪酰胺开始制备生物素化酪胺。要制备 10 mM 储备溶液，请将 3.6 mg 生物素-酪胺溶解在 1 mL DMSO 中。将溶解的产品等分储存在-20°C以保持质量。</li>
</ol>2. 维持培养中的HT-29细胞
注意：表达ZBP1的HT-29是通过用编码人ZBP1的lentivector27 转导产生的。
<ol><li>将表达ZBP1的HT-29细胞保持?...

<ol>
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该免疫荧光染色方案描述了使用酪胺信号扩增（TSA）来提高难以检测的人类坏死性凋亡信号通路信号事件的灵敏度，包括RIPK3和MLKL26的磷酸化。TSA步骤的加入显著提高了p-RIPK3（S227）和p-MLKL（S358）的检测阈值，并提高了p-MLKL（S358）应变的灵敏度。TSA显示模拟处理的样品中已经存在p-RIPK3（S227）信号。在人细胞中，RIPK3在S227处的自磷酸化是通过与MLKL稳定相互作用来激活坏死性凋亡?...

受体相互作用的丝氨酸/苏氨酸蛋白激酶 3 （RIPK3） 和混合谱系激酶结构域样 （MLKL） 是坏死性凋亡细胞死亡的中枢调节因子1，2。坏死性凋亡是一种溶解性和炎症性调节性细胞死亡形式，涉及抗病毒免疫和自身炎症。病毒感染细胞的坏死性凋亡会立即关闭病毒复制。坏死性凋亡诱导后的细胞裂解也会释放与损伤相关的分子模式，从而刺激抗病毒免疫3，4。坏死性凋亡是由 RIP 同型相互作用基序 （RHIM） 介导的与三种上游活化分子之一的相互作用后激活 RIPK3 引发的：RIPK1（TNF 受体 1 [TNFR1] 接合时）、含 TIR 结构域的适配器诱导干扰素β（TRIF;在 Toll 样受体 3 和 4 接合时）或抗病毒核酸传感器 Z-DNA 结合蛋白 1 （ZBP1）1，2.坏死性凋亡信号传导通过一系列磷酸化事件进行，从RIPK3的自磷酸化开始。人RIPK3在其激酶结构域内的丝氨酸（S）227处的自磷酸化是通过与MLKL相互作用来使坏死性凋亡的先决条件，并且通常用作人RIPK3活化和坏死性凋亡细胞死亡的生化标志物1，5。一旦激活，RIPK3在苏氨酸（T）357和S3581处磷酸化MLKL的活化环。这会导致MLKL构象发生变化，导致N末端四螺旋束结构域暴露。然后MLKL寡聚并运输到细胞膜，在那里它通过在脂质双层中插入暴露的四个螺旋束形成孔，最终导致细胞死亡2，6。
ZBP1 是一种抗病毒核酸传感器，可识别左旋 Z 型核酸，包括 Z 构象 （Z-RNA） 中的双链 RNA。Z-RNA结合通过位于ZBP1的N末端的两个Zα结构域发生。在RNA和DNA病毒感染过程中积累的Z-RNA被认为直接参与ZBP17，8。活化的ZBP1通过其中枢RIM募集RIPK3并诱导调节细胞死亡，包括坏死性凋亡9，10。病毒已经采用了许多逃逸机制来抵消ZBP1诱导的宿主细胞坏死性凋亡11。例如，单纯疱疹病毒 1 （HSV-1） 核糖核苷酸还原酶亚基 1，称为 ICP6 并由 UL39 编码，在其 N 末端含有 RHIM，干扰人细胞中 ZBP1 介导的 RIPK3 活化12，13，14，15。ZBP1不仅限制病毒复制，而且小鼠研究表明，ZBP1激活引起炎症性疾病并刺激癌症免疫16，17，18，19，20，21。因此，检测ZBP1诱导的人类细胞坏死性凋亡期间发生的信号事件的协议对于评估ZBP1在这些过程中的作用很有价值。
酪胺信号放大（TSA），也称为催化报告沉积（CARD），已被开发用于提高基于抗体的免疫测定中的检测限和信噪比。在TSA期间，任何一抗都可用于检测目标抗原。辣根过氧化物酶（HRP）与二抗偶联，在过氧化氢存在下催化生物素化酪胺自由基的局部积聚。这些活化的生物素-酪胺自由基然后与近端酪氨酸残基反应形成共价键。潜在的酪胺-生物素底物包括抗原本身、一抗和二抗以及邻近的蛋白质。因此，虽然TSA显着提高了测定的灵敏度，但其一些空间分辨率会丢失。在最后一步中，使用荧光标记的链霉亲和素检测生物素分子。HRP反应在目标抗原上或附近沉积了许多酪酰胺-生物素分子。这大大增加了链霉亲和素-荧光染料结合位点的数量，从而大大增强了测定的灵敏度（图1）。或者，酪胺可以直接与荧光染料偶联，无需链霉亲和素偶联的荧光团。蛋白质免疫组织化学和DNA/RNA 原位 杂交是最早采用TSA提高信号强度的方法之一22，23。最近，TSA已与细胞内流式细胞术24 和质谱术25相结合。
在这里，我们提出了一种方案，用于检测丝氨酸227磷酸化人RIPK3（p-RIPK3 [S227]）和磷酸化人MLKL（p-MLKL [S358]）在HSV-1感染激活ZBP1时使用免疫荧光显微镜。我们使用坏死性凋亡敏感的HT-29人结直肠腺癌细胞系，该细胞系被转导以稳定表达人ZBP1。这些细胞感染了表达突变ICP6蛋白（HSV-1 ICP6mutRHIM）的HSV-1菌株，其中病毒RHIM（VQCG）内的四个核心氨基酸被丙氨酸（AAAA）取代，从而使ICP6无法阻断ZBP1介导的坏死性凋亡13，14，15。为了克服目前市售的针对p-RIPK3和p-MLKL的抗体在免疫染色26中的低信噪比问题，我们执行了酪胺信号放大（TSA）步骤（图1），该步骤可对人p-RIPK3（S227）进行稳健检测，并将人p-MLKL（S358）的检测灵敏度提高一个数量级。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rabbit HRP</td>
			<td>Agilent Technologies Belgium</td>
			<td>K4002</td>
			<td>Envision+ System-HRP Labelled Polymer anti-rabbit</td>
		</tr>
		<tr>
			<td>Goat anti-mouse DyLight 633</td>
			<td>Thermofisher</td>
			<td>35513</td>
			<td>Secundary antibody</td>
		</tr>
		<tr>
			<td>HSV-1 ICP0</td>
			<td>Santa Cruz</td>
			<td>sc-53070</td>
			<td>Mouse anti-ICP0(HSV-1) antibody</td>
		</tr>
		<tr>
			<td>IAV-PR8 mouse serum</td>
			<td>In house production</td>
			<td>xx</td>
			<td>Mouse anti-IAV-PR8 polyclonal antibody</td>
		</tr>
		<tr>
			<td>pMLKL</td>
			<td>Abcam</td>
			<td>ab187091</td>
			<td>Rabbit anti-MLKL-phospho S358 antibody</td>
		</tr>
		<tr>
			<td>pRIPK3</td>
			<td>Abcam</td>
			<td>ab209384</td>
			<td>Rabbit anti-RIPK3-phospho S227 antibody</td>
		</tr>
		<tr>
			<td>Fluorophores</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermofisher</td>
			<td>D21490</td>
			<td>To visualise the nucleus of the cells</td>
		</tr>
		<tr>
			<td>Streptavidin coupled to Alexa Fluor 568</td>
			<td>Thermofisher</td>
			<td>S11226</td>
			<td>To visulalise biotin molecules</td>
		</tr>
		<tr>
			<td>Compounds</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30% H2O2</td>
			<td>Sigma</td>
			<td>H1009</td>
			<td>Oxidising substrate, necessary for HRP activity</td>
		</tr>
		<tr>
			<td>4% PFA</td>
			<td>SANBIO</td>
			<td>AR1068</td>
			<td>To fix/crosslink the cells</td>
		</tr>
		<tr>
			<td>Biotinyl-tyramide</td>
			<td>R&#38;D Systems</td>
			<td>6241</td>
			<td>To amplify signal, HRP substrate</td>
		</tr>
		<tr>
			<td>BV-6</td>
			<td>Selleckchem</td>
			<td>S7597</td>
			<td>BV6 IAP Inhibitor</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>For cell culture: to detach the cells</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>8.0 g/L NaCl</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>0.4 g/L disodium salt of EDTA</td>
		</tr>
		<tr>
			<td>EDTA 0.04%</td>
			<td>In house formulation</td>
			<td></td>
			<td>1.1 g/L Na2HPO4</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>0.2 g/L NaH2PO4</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>0.2 g/L KCl</td>
		</tr>
		<tr>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>&#160;&#160;</td>
			<td>0.2 g/L Glucose</td>
		</tr>
		<tr>
			<td>Fetal Bovine serum</td>
			<td>TICO</td>
			<td>FBS EU XXX</td>
			<td>For cell culture, maintaining cell culture; lot number: 90439</td>
		</tr>
		<tr>
			<td>GSK&#39;840</td>
			<td>Aobious</td>
			<td>AOB0917</td>
			<td>RIPK3 kinase inhibitor</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>G7513</td>
			<td>For cell culture, maintaining cell culture</td>
		</tr>
		<tr>
			<td>MAXblock</td>
			<td>Active Motif</td>
			<td>15252</td>
			<td>Blocking solution</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10444402</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>S8636</td>
			<td>For cell culture, maintaining cell culture</td>
		</tr>
		<tr>
			<td>TNF-&#945;</td>
			<td>In house production</td>
			<td>-</td>
			<td>Signaling molecule, able to trigger cell death in combination with BV6 and zVAD</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>T8787-50ML</td>
			<td>To permeabilise the cells</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Merck</td>
			<td>11732</td>
			<td>For cell counting, used as live/dead marker at 0,1%</td>
		</tr>
		<tr>
			<td>Trypsine</td>
			<td>Sigma-Aldrich</td>
			<td>T4424</td>
			<td>For cell culture: to detach the cells</td>
		</tr>
		<tr>
			<td>zVAD</td>
			<td>Bachem</td>
			<td>BACE4026865.0005</td>
			<td>Z-Val-Ala-DL-Asp-fluoromethylketone</td>
		</tr>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#181;-Slide 8 well high glass bottom</td>
			<td>iBidi</td>
			<td>80807</td>
			<td>To culture the cells</td>
		</tr>
		<tr>
			<td>Cotton Preping Balls-size medium</td>
			<td>Electron Microscopy Sciences</td>
			<td>71001-10</td>
			<td>To clean the objectives</td>
		</tr>
		<tr>
			<td>Immersol 518 F / 30 &#176;C</td>
			<td>ZEISS</td>
			<td>444970-9000-000</td>
			<td>To visualise the sample at high magnifications</td>
		</tr>
		<tr>
			<td>Lens Cleaner</td>
			<td>ZEISS</td>
			<td>000000-0105-200</td>
			<td>To clean the objectives</td>
		</tr>
		<tr>
			<td>LSM880 Fast Airyscan confocal microscope</td>
			<td></td>
			<td></td>
			<td>To visualise the sample</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Office</td>
			<td>xx</td>
			<td>To process the data</td>
		</tr>
		<tr>
			<td>Prism 9</td>
			<td>Graphpad</td>
			<td>xx</td>
			<td>To analyse the data- statistical testing and graph generation</td>
		</tr>
		<tr>
			<td>Volocity 6.3</td>
			<td>Volocity</td>
			<td>xx</td>
			<td>To perform quantifications</td>
		</tr>
		<tr>
			<td>Zen black</td>
			<td>ZEISS</td>
			<td>xx</td>
			<td>To aquire and process images</td>
		</tr>
		<tr>
			<td>Zen blue</td>
			<td>ZEISS</td>
			<td>xx</td>
			<td>To visualise images</td>
		</tr>
		<tr>
			<td>Viruses</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HSV-1 (mutRHIM) F strain</td>
			<td>produced by&#160; Dr. Jiahuai Han</td>
			<td>in house replication</td>
			<td>HSV-1 as a trigger for necroptosis; RHIM core domain of UL39/ICP6 is mutated (VQCG&#62;AAAA)</td>
		</tr>
		<tr>
			<td>HSV-1 (WT) F strain</td>
			<td>Produced by Dr. Jiahuai Han</td>
			<td>in house replication</td>
			<td>HSV-1 (WT) as a negative control for necroptosis induction (ICP6 inhibition)</td>
		</tr>
		<tr>
			<td>IAV PR8</td>
			<td>in house stock</td>
			<td>in house replication</td>
			<td>IAV as a trigger for necroptosis</td>
		</tr>
	</tbody>
</table>

tyramide signal amplification for the immunofluorescent staining of zbp1-dependent phosphorylation of ripk3 and mlkl after hsv-1 infection in human cells

激酶受体相互作用丝氨酸/苏氨酸蛋白激酶3（RIPK3）及其底物混合谱系激酶结构域样（MLKL）是坏死性凋亡的关键调节因子，坏死性凋亡是一种具有重要抗病毒功能的细胞死亡的炎症形式。RIPK3的自磷酸化诱导坏死性凋亡MLKL的孔形成刽子蛋白的磷酸化和活化。细胞膜上磷酸化MLKL的运输和寡聚化导致细胞裂解，这是坏死性凋亡细胞死亡的特征。核酸传感器ZBP1在感染RNA和DNA病毒后通过与左旋Z型双链RNA（Z-RNA）结合来激活。ZBP1活化通过诱导受感染宿主细胞的调节细胞死亡（包括坏死性凋亡）来限制病毒感染。免疫荧光显微镜允许在每个细胞的基础上可视化ZBP1介导的坏死性凋亡下游的不同信号传导步骤。然而，使用目前市售的针对人RIPK3和MLKL的磷酸化特异性抗体的标准荧光显微镜的灵敏度排除了这些标志物的可重复成像。在这里，我们描述了感染单纯疱疹病毒1（HSV-1）的人HT-29细胞中丝氨酸（S）磷酸化RIPK3（S227）和MLKL（S358）的优化染色程序。在免疫荧光染色方案中加入酪胺信号放大（TSA）步骤可以特异性检测S227磷酸化的RIPK3。此外，TSA大大提高了S358磷酸化MLKL检测的灵敏度。总之，该方法能够在诱导ZBP1诱导的坏死性凋亡期间可视化这两个关键信号事件。

人体细胞中MLKL磷酸化，尤其是RIPK3磷酸化的免疫荧光检测在技术上具有挑战性26。我们在这里提出了一种改进的ZBP1激活后人p-RIPK3（S227）和p-MLKL（S358）染色方案。该协议包括一个TSA步骤，以提高荧光信号的检测限和灵敏度。为了验证该方法，对TSA介导的免疫荧光与p-RIPK3（S227）和p-MLKL（S358）的标准间接荧光染色进行了并排比较。
用ICP6 RHIM突变型HSV-1菌株（HSV...

Watch this Scientific Journal Video about Tyramide Signal Amplification for the Immunofluorescent Staining of ZBP1-Dependent Phosphorylation of RIPK3 and MLKL After HSV-1 Infection in Human Cells at J

Tyramide Signal Amplification for the Immunofluorescent Staining of ZBP1-Dependent Phosphorylation of RIPK3 and MLKL After HSV-1 Infection in Human Cells

免疫荧光染色期间的酪胺信号扩增能够在 HSV-1 感染后 ZBP1 诱导的坏死性凋亡期间灵敏地检测磷酸化的 RIPK3 和 MLKL。

人细胞中HSV-1感染后RIPK3和MLKL的ZBP1依赖性磷酸化的免疫荧光荧光放大的酪胺信号扩增

The kinase Receptor-interacting serine/threonine protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like (MLKL) are critical ...

磷酸化RIPK3和MLKL的稳健可视化是坏死性凋亡诱导过程中的两个关键信号事件，在技术上具有挑战性。所提出的酪酰胺扩增方案能够灵敏地检测这两种分子。信号放大步骤降低了检测阈值，允许对磷酸化的RIPK3和MLKL进行稳健且可重复的检测。开始将 90， 000 个表达 ZBP1 的 HT-29 细胞接种在全强度 McCoy 的 5A 培养基中，置于 1 厘米见方表面积的孔板中，允许高端显微镜检查。每孔使用200微升的结束体积。将细胞在37摄氏度下用5%二氧化碳孵育过夜。一旦细胞达到70%至80%汇合度，在含有单纯疱疹病毒一的200微升预热的全强度McCoy's 5A培养基中感染细胞，以5的感染倍数编码边缘突变体ICP6。将细胞与病毒孵育九小时以诱导坏死性凋亡。作为坏死性凋亡诱导的阳性对照，用200微升预热的坏死性凋亡诱导鸡尾酒刺激细胞四小时。作为阴性对照，加入22微升10微摩尔GSK840预热37摄氏度以抑制RIPK3激酶活性。在未经治疗的条件下和坏死性凋亡刺激后包括该抑制剂，以防止丝氨酸227的自磷酸化。要固定细胞，请取出培养基并用200微升PBS洗涤细胞。然后取出PBS，加入150微升在室温下平衡的4%多聚甲醛。将细胞在室温下孵育30分钟。固定后，除去4%多聚甲醛，用200微升PBS洗涤细胞三次。样品可以在超过PBS的4摄氏度下储存过夜，直到进一步处理。从平板上取出PBS，并在室温下将细胞与100微升渗透缓冲液在倾斜的实验室振荡器上孵育30分钟。孵育后，除去透化缓冲液，并用100微升洗涤缓冲液洗涤细胞。将成像室与洗涤缓冲液在室温下在倾斜的实验室摇床上孵育五分钟。为防止一抗的非特异性结合，请将板与100微升封闭培养基在室温下在倾斜的实验室摇床上孵育两小时。接下来，用100微升洗涤缓冲液洗涤孔三次，在室温下在倾斜的实验室摇床上孵育五分钟。除去洗涤缓冲液后，向成像室中加入100微升一抗。确保包括无一抗条件，以可视化酪胺信号放大的潜在背景，并设置掩蔽阈值。将腔室在四摄氏度下孵育过夜。除去一抗混合物，并用100微升洗涤缓冲液洗涤孔三次，在室温下在倾斜的实验室摇床上孵育5分钟。去除洗涤缓冲液后，用100微升HRP标记的二抗孵育细胞，识别要在倾斜的实验室摇床上扩增30分钟的一抗种类。在抗体孵育期间，制备生物素化酪胺混合物。为了触发HRP的酶活性，请在使用前将氧化底物新鲜添加到TSA缓冲液中。然后使用优化的稀释液在制备的TSA缓冲液中稀释生物素化的酪胺。用HRP标记的二抗孵育后，用洗涤缓冲液洗涤孔三次，在倾斜的实验室摇床上孵育五分钟。从孔中取出洗涤缓冲液后，向孔中加入稀释的生物素 - 酪胺至100微升的结束体积。在室温下在倾斜的实验室摇床上孵育反应10分钟，然后进行三次洗涤缓冲液洗涤。制备含有二抗、偶联链霉亲和素和核染色和洗涤缓冲液的染色混合物。取出洗涤缓冲液，并在室温下将板与100微升染色混合物在倾斜的实验室摇床上孵育一小时。从此步骤开始，保持成像室免受光线照射。染色后，用洗涤缓冲液连续洗涤两次，并用PBS冲洗孔两次。将超过PBS的样品储存在4摄氏度下，以保存染色直至成像。成像室现在可以在共聚焦显微镜上可视化。酪胺信号扩增的加入能够可靠地检测编码边缘突变体ICP6感染细胞的单纯疱疹病毒1的细胞质中丝氨酸227处磷酸化的RIPK3。2%的激光功率足以进行灵敏检测。在标准的间接免疫荧光中，较高的激光功率仍然不足以检测磷酸化-RIPK3。在三维Z-stack图像的定量中，感染细胞显示磷酸化RIPK3阳性体素比模拟处理的细胞增加约20倍。与模拟处理的细胞相比，野生型单纯疱疹病毒一感染细胞的磷酸化RIPK3染色增加表明ICP6的边缘结构域无法完全阻断丝氨酸227处的RIPK3磷酸化。GSK840与RIPK3的激酶结构域结合，并在ZBP1激活时阻止丝氨酸227处的RIPK3磷酸化，证实了酪胺信号放大介导的磷酸化RIPK3检测方法的特异性。模拟处理的细胞在丝氨酸358处的MLKL磷酸化略微间断的胞质染色较低，而在感染细胞的细胞质，细胞核和质膜中检测到强染色。标准的间接免疫荧光需要40%的激光功率来特异性检测感染细胞中的磷酸化MLKL信号。相比之下，酪胺信号放大提高了磷酸化MLKL的检测灵敏度，从而将必要的激光功率降低到6%3D Z-stack图像量化显示，与使用标准方法相比，使用酪胺信号放大的磷酸化MLKL阳性体素数量增加了约90倍，这表明磷酸化MLKL的检测阈值和灵敏度有所提高。与单纯疱疹病毒类似，甲型流感病毒感染引发ZBP1依赖性坏死性凋亡。酪胺信号放大允许对磷酸化-RIPK3和磷酸化-MLKL进行稳健的检测，表明坏死性凋亡。为了解释共聚焦染色，需要包括几个染色对照。这需要无原发条件、单一染色以及阳性对照。通过调整该协议，可以使用顺序TSA扩增多个靶标。这将允许在同一细胞内检测磷酸化的RIPK3和MLKL。对坏死性凋亡生物标志物的灵敏检测对于正在进行的关于自身炎症或病毒感染的ZBP1研究具有洞察力，在这些研究中，执行的ZBP1依赖性细胞死亡途径仍需要确认为坏死性凋亡，细胞凋亡或焦亡。

Research

JoVE Journal

Immunology and Infection

Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

协同刺激信号封锁的同种抗原刺激诱导人外周血单个核细胞同种异体抗原特异性无能

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic ...

科研

免疫与感染

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

选择恶性疟原虫</emCytoadhesion>寄生虫对人脑血管内皮细胞

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

Following Cell-fate in E. coli After Infection by Phage Lambda

Following Cell-fate in E. coli After Infection by Phage Lambda

在细胞命运<em&gt; E.大肠杆菌</em感染后的噬菌体λ

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac转座子初级人类T细胞系统改造

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

与碘酸雪夫染色检测糖原的外周血单个核细胞

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

冻干环介导等温扩增试剂的检测中的发展<em&gt;贝氏柯克斯体</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

人精切肺切片中物质的细胞毒性及免疫调节作用的评价

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

单细胞荧光显微镜定量 Efferocytosis 的研究

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue

脂肪组织中的血管和神经纤维的共染色

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of ...

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

恒流中原发性人类内皮细胞的肺炎球菌感染

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...