Name: fluorescence-activated cell sorting-radioligand treated tissue (facs-rtt) to determine the cellular origin of radioactive signal
Uploaded: 2021-09-10T14:30:00
Description: Watch this Scientific Journal Video about Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue FACS-RTT to Determine the Cellular Origin of Radioactive Signal at JoVE.com

这项工作得到了瑞士国家科学基金会的支持（拨款320030-184713）。作者BBT和KC得到了Velux基金会（项目号1123）的支持。作者ST获得了瑞士国家科学基金会的支持（早期博士后流动奖学金，No.P2GEP3_191446），Max Cloetta教授基金会（临床医学加奖学金）和Jean和Madeleine Vachoux基金会。

所有实验程序均分别与日内瓦州人类和动物实验伦理委员会，州研究伦理委员会（CCER）和日内瓦州（瑞士）健康总方向达成协议。数据按照动物研究报告：报告 体内 实验（ARRIVE）指南。
1. SPECT相机的准备和校准
<ol><li>打开相机，加载操作软件（参见 材料表）。单击“ 主 XYZ 舞台 ”按钮以执行归位。</li>
	<li>设置由一个10分钟的扫描采集组成...

<ol>
	<li>Nichols, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=regional,+and+national+burden+of+Alzheimer's+disease+and+other+dementias,+1990-2016:+a+systematic+analysis+for+the+Global+Burden+of+Disease+Study+2016.">regional, and national burden of Alzheimer's disease and other dementias, 1990-2016: a systematic analysis for the Global Burden of Disease Study 2016.</a> The Lancet Neurology. 18 (1), 88-106 (2019).</li><li>Kinney, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+as+a+central+mechanism+in+Alzheimer's+disease.">Inflammation as a central mechanism in Alzheimer's disease.</a> Alzheimer's &amp; Dementia. 4, 575-590 (2018).</li><li>D'Amelio, M., Puglisi-Allegra, S., Mercuri, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+dopaminergic+midbrain+in+Alzheimer's+disease:+Translating+basic+science+into+clinical+practice.">The role of dopaminergic midbrain in Alzheimer's disease: Translating basic science into clinical practice.</a> Pharmacological Research. 130, 414-419 (2018).</li><li>D'Amelio, M., Serra, L., Bozzali, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventral+tegmental+area+in+prodromal+Alzheimer's+disease:+Bridging+the+gap+between+mice+and+humans.">Ventral tegmental area in prodromal Alzheimer's disease: Bridging the gap between mice and humans.</a> Journal of Alzheimer's Disease: JAD. 63 (1), 181-183 (2018).</li><li>Cohen, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transgenic+Alzheimer+rat+with+plaques,+tau+pathology,+behavioral+impairment,+oligomeric+a&#946;,+and+frank+neuronal+loss.">A transgenic Alzheimer rat with plaques, tau pathology, behavioral impairment, oligomeric a&#946;, and frank neuronal loss.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 33 (15), 6245-6256 (2013).</li><li>Morrone, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+differences+in+Alzheimer's+disease+pathology+confound+behavioural+rescue+after+amyloid-&#946;+attenuation.">Regional differences in Alzheimer's disease pathology confound behavioural rescue after amyloid-&#946; attenuation.</a> Brain: A Journal of Neurology. 143 (1), 359-373 (2020).</li><li>Berkowitz, L. E., Harvey, R. E., Drake, E., Thompson, S. M., Clark, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+impairment+of+directional+and+spatially+precise+trajectories+by+TgF344-Alzheimer's+disease+rats+in+the+Morris+Water+Task.">Progressive impairment of directional and spatially precise trajectories by TgF344-Alzheimer's disease rats in the Morris Water Task.</a> Scientific Reports. 8 (1), 16153 (2018).</li><li>Koulousakis, P., vanden Hove, D., Visser-Vandewalle, V., Sesia, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+improvements+after+intermittent+deep+brain+stimulation+of+the+nucleus+basalis+of+meynert+in+a+transgenic+rat+model+for+Alzheimer's+disease:+A+preliminary+approach.">Cognitive improvements after intermittent deep brain stimulation of the nucleus basalis of meynert in a transgenic rat model for Alzheimer's disease: A preliminary approach.</a> Journal of Alzheimer's Disease: JAD. 73 (2), 461-466 (2020).</li><li>Tournier, B. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+reference+learning+deficits+in+absence+of+dysfunctional+working+memory+in+the+TgF344-AD+rat+model+of+Alzheimer's+disease.">Spatial reference learning deficits in absence of dysfunctional working memory in the TgF344-AD rat model of Alzheimer's disease.</a> Genes, Brain, and Behavior. , 12712 (2020).</li><li>Tournier, B. B., Tsartsalis, S., Ceyz&#233;riat, K., Garibotto, V., Millet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+TSPO+signal+and+neuroinflammation+in+Alzheimer's+disease.">In vivo TSPO signal and neuroinflammation in Alzheimer's disease.</a> Cells. 9 (9), (2020).</li><li>Backes, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[11C]raclopride+and+extrastriatal+binding+to+D2/3+receptors.">[11C]raclopride and extrastriatal binding to D2/3 receptors.</a> NeuroImage. 207, 116346 (2020).</li><li>Millet, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+dopamine+D(2/3)+receptors+in+rat+brain+using+factor+analysis+corrected+[18F]Fallypride+images.">Quantification of dopamine D(2/3) receptors in rat brain using factor analysis corrected [18F]Fallypride images.</a> NeuroImage. 62 (3), 1455-1468 (2012).</li><li>Tsartsalis, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+simplified+reference+tissue+model+for+the+quantification+of+dopamine+D2/3+receptors+with+[18F]Fallypride+images.">A modified simplified reference tissue model for the quantification of dopamine D2/3 receptors with [18F]Fallypride images.</a> Molecular Imaging. 13 (8), (2014).</li><li>Schwarz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+fluorescence-activated+cell+sorting+to+examine+cell-type-specific+gene+expression+in+rat+brain+tissue.">Using fluorescence-activated cell sorting to examine cell-type-specific gene expression in rat brain tissue.</a> Journal of Visualized Experiments: JoVE. (99), e52537 (2015).</li><li>Tournier, B. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-activated+cell+sorting+to+reveal+the+cell+origin+of+radioligand+binding.">Fluorescence-activated cell sorting to reveal the cell origin of radioligand binding.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 40 (6), 1242-1255 (2020).</li><li>Tournier, B. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytic+TSPO+upregulation+appears+before+microglial+TSPO+in+Alzheimer's+disease.">Astrocytic TSPO upregulation appears before microglial TSPO in Alzheimer's disease.</a> Journal of Alzheimer's Disease: JAD. 77 (3), 1043-1056 (2020).</li></ol>

据我们所知，该技术是第一个描述一种方法的方法，该方法可以更好地了解细胞水平上放射性示踪剂的体内结合改变。该协议描述了一种多尺度方法，以[125 I]CLINDE（TSPO）或[125I]R91150（5HT2AR）为例，在细胞水平上量化放射性示踪剂结合。
该技术足够强大且灵敏，可以精确检测广谱神经胶质细胞改变的细胞起源，从LPS诱导的强烈炎症反应到AD大鼠?...

神经退行性疾病，如阿尔茨海默病（AD），其特征在于与症状增加相关的神经元丢失。AD是痴呆症最常见的原因，占病例的60%-70%，影响全球约5000万人1。在神经病理学水平上，AD的两个主要特征是细胞外淀粉样蛋白β（Aβ）斑块和细胞内Tau神经原纤维缠结的积累。神经胶质细胞改变也与AD2 和几种神经递质系统的可能破坏有关3，4。
TgF344-AD大鼠品系通过表达人APP和PS1ΔE9 转基因来模拟AD，导致可溶性和不溶性Aβ-40和Aβ-42表达以及淀粉样蛋白斑块形成5。它还呈现Tau蛋白的高磷酸化形式的积累，导致tau病。从9-24个月大，大鼠逐渐发展AD的病理标志和认知障碍5，6，7，8，9。
正电子发射断层扫描（PET），单光子发射计算机断层扫描（SPECT）和放射自显影是基于γ射线发射和定量的技术。放射性示踪剂在体内（PET和SPECT）或离体/体外（放射自显影）进行定量。这些敏感的技术有助于理解几种脑部疾病的机制，如AD。事实上，在神经炎症方面，有很多研究评估18 kDa易位蛋白（TSPO），一种体内神经炎症标志物，放射性标记的示踪剂，如[11C]-（R）-PK11195或[11C]PBR28（综述见10）。此外，已经使用放射性示踪剂11，12，13研究了神经递质系统的改变。
然而，这些技术并不能确定放射性信号的细胞来源。这可能会妨碍对PET / SPECT中放射性配体结合改变的生物学基础的解释。例如，在TSPO神经炎症研究的情况下，了解TSPO的增加或减少是由于星细胞还是小胶质细胞的变化至关重要。荧光活化细胞分选为放射性配体处理组织（FACS-RTT）技术是为了解决这些问题而开发的，允许分别评估每种细胞类型中的放射性配体结合并量化每个细胞的靶蛋白密度。因此，这种创新技术与PET和SPECT成像相辅相成且高度兼容。
在这里，该技术沿两个轴应用：使用TSPO特异性放射性配体研究神经炎症和评估5-羟色胺能系统。在第一个轴上，目的是了解TSPO信号响应急性炎症反应的细胞起源。因此，在通过脂多糖（LPS）注射诱导神经炎症后，以及在 体内 [125I]CLINDE SPECT成像研究之后，FACS-RTT用于大鼠的脑组织。此外，对12个月和24个月大的TgF344-AD大鼠和匹配的野生型（WT）大鼠应用相同的成像和FACS-RTT方案。第二轴旨在通过按细胞类型 进行离体 5-HT2AR密度评估来确定该大鼠模型中5-羟色胺能系统改变的起源。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BioVet</td>
			<td>BioVet</td>
			<td></td>
			<td>Software for vitals check</td>
		</tr>
		<tr>
			<td>Bondclone C18 reverse-phase column</td>
			<td>Phenomenex, Schlieren, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Des-Sur</td>
			<td>University Hospital of Geneva</td>
			<td></td>
			<td>Virucide</td>
		</tr>
		<tr>
			<td>Fc Block / anti-CD32</td>
			<td>BD Biosciences</td>
			<td>BDB550270</td>
			<td>Reactivity for rat</td>
		</tr>
		<tr>
			<td>FITC-conjugated anti-rat CD90</td>
			<td>Biolegend</td>
			<td>202504</td>
			<td>Reactivity for rat</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>B. Braun</td>
			<td>B01AB01</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC</td>
			<td>Knauer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insyte-W 24 GA 0.75 IN 0.7 x 19 mm</td>
			<td>BD Biosciences</td>
			<td>321312</td>
			<td>24 G catheter</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>ZDG9623</td>
			<td></td>
		</tr>
		<tr>
			<td>Lacryvisc</td>
			<td>Alcon</td>
			<td>2160699</td>
			<td></td>
		</tr>
		<tr>
			<td>LS Columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS MultiStand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropore soft tape</td>
			<td>3M</td>
			<td>F51DA01</td>
			<td></td>
		</tr>
		<tr>
			<td>MILabs-Uspect II</td>
			<td>MILabs</td>
			<td></td>
			<td>Software for SPECT Camera</td>
		</tr>
		<tr>
			<td>MoFlo Astrios</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>Cell sorter</td>
		</tr>
		<tr>
			<td>Myelin Removal Beads II</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-733</td>
			<td>Contains beads and myelin removal buffer.</td>
		</tr>
		<tr>
			<td>NaCl 0.9% Sterile solution</td>
			<td>B. Braun</td>
			<td>395202</td>
			<td></td>
		</tr>
		<tr>
			<td>Neural Dissociation Kit (P)</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-628</td>
			<td>Contains the enzyme mixes, pipets 1, 2 and 3.</td>
		</tr>
		<tr>
			<td>Nylon Mesh Sheet</td>
			<td>Amazon</td>
			<td>CMN-0074-10YD</td>
			<td>40 inch width, 80 micron size mesh</td>
		</tr>
		<tr>
			<td>Peracetic acid</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QuadroMACS Separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-976</td>
			<td></td>
		</tr>
		<tr>
			<td>R91150 pr&#233;cursor</td>
			<td>CERMN</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sep-Pak C18 Column</td>
			<td>Waters</td>
			<td></td>
			<td>Concentration column</td>
		</tr>
		<tr>
			<td>Sodium iodide Na125</td>
			<td>PerkinElmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tributylin precursor</td>
			<td>CERMN</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>U-SPECT Rec2.38c</td>
			<td>MILabs</td>
			<td>Version Rec2.38c</td>
			<td>Software for SPECT images reconstruction</td>
		</tr>
		<tr>
			<td>USPECT II</td>
			<td>MILabs</td>
			<td></td>
			<td>Spect Camera</td>
		</tr>
		<tr>
			<td>Wizard 3&#34;</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Gamma counter</td>
		</tr>
	</tbody>
</table>

fluorescence-activated cell sorting-radioligand treated tissue (facs-rtt) to determine the cellular origin of radioactive signal

神经胶质细胞可能在神经退行性疾病（如阿尔茨海默病（AD））的病理生理学中具有相当大的影响。它们的改变可能与促炎状态有关。TgF344-AD大鼠菌株设计用于表达人类APP和人类PS1ΔE9 基因，编码淀粉样蛋白Aβ-40和Aβ-42，并随着年龄的增长显示淀粉样蛋白病理学和认知缺陷。本研究使用TgF344-AD大鼠模型来评估18 kDa易位蛋白（TSPO，神经胶质细胞活化的标志物）结合的细胞起源，以及可能在AD中破坏的5HT2A受体（5HT2AR）血清素受体水平。这里介绍的技术是荧光活化细胞分选为放射性配体处理组织（FACS-RTT），这是一种定量细胞类型特异性技术，补充 了体内 PET或SPECT或 离体/体外 放射自显影技术。它在细胞术细胞分选后使用γ计数器量化先前用于成像的相同放射性标记示踪剂。这允许确定具有高细胞特异性和灵敏度的放射性标记蛋白的细胞来源。例如，FACS-RTT的研究表明，（i）脂多糖（LPS）诱导的神经炎症大鼠模型中TSPO结合的增加与小胶质细胞相关，（ii）12个月和18个月时TSPO结合的增加首先与星形胶质细胞相关，然后与野生型（WT）大鼠相比，TgF344-AD大鼠中的小胶质细胞相关，以及（iii）纹状体密度为5HT2A在同一大鼠AD模型中，R在18个月时星形胶质细胞减少。有趣的是，这种技术可以扩展到几乎所有的放射性示踪剂。

WT大鼠在单侧LPS注射后使用[125I]CLINDE放射性示踪剂进行体内SPECT扫描（图2）。该扫描（使用放射性示踪剂注射后45-60分钟图像的汇总数据）显示LPS注射部位[125I]CLINDE的结合（图2A）高于大脑对侧区域（图2B）。接受FACS-RTT的离体样品证实了这些结果，并显示仅在小胶质细胞中存在较多的[125I]CLINDE...

Watch this Scientific Journal Video about Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue FACS-RTT to Determine the Cellular Origin of Radioactive Signal at JoVE.com

Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue FACS-RTT to Determine the Cellular Origin of Radioactive Signal

荧光激活细胞分选 - 放射性配体处理组织（FACS-RTT）是研究18 kDa易位蛋白或血清素5HT2A受体表达在细胞尺度上阿尔茨海默病中的作用的强大工具。该协议描述了FACS-RTT在TgF344-AD大鼠模型中的 离体 应用。

荧光活化细胞分选-放射性配体处理组织（FACS-RTT）以确定放射性信号的细胞来源

Glial cells probably have a considerable implication in the pathophysiology of neurodegenerative disorders, such as Alzheimer's disease (AD). Their ...

该方案是对体内成像的补充。它允许放射性示踪剂的细胞类型和细胞水平定量，这与在体内获得的量表不同。该技术的主要优点是其灵敏度和通过使用放射性配体和细胞分选给出的细胞分辨率。打开照相机并加载操作软件。单击“主 XYZ”阶段按钮以执行归位。设置由一个 10 分钟的扫描采集组成的实验。将“步进”模式设置为“精细”，将“采集”模式设置为“列表”模式以记录整个发射光谱。设置床，确保加温系统，呼吸传感器和麻醉功能正常且安全。然后，在动物头部的位置放置一个幻影。通过在屏幕底部的三个图像的帮助下滑动三个维度的光标来设置扫描区域。确保幻像和动物的扫描体积相同。启动幻像扫描，以便使用先前建立的参数进行后续校准。确保在适当的环境中工作，授权用于涉及放射性的实验。将100微克三丁基素前体与碘化钠和5微升37%对乙酰乙酸在70摄氏度下在手套箱中的热循环器中孵育20分钟。使用50%乙腈在水中稀释反应，以达到500微升的体积。将500微升稀释的反应注入反相柱上。用线性梯度HPLC分离CLINDE，在7毫摩尔磷酸中运行从5%至95%ACN运行10分钟。将分离物在水中稀释以达到10毫升的最终体积，然后将稀释的反应注入浓缩柱上。用300微升无水乙醇从柱中洗脱CLINDE，然后通过在室温下在真空离心机中孵育乙醇40分钟来蒸发乙醇。将含有CLINDE的残留物稀释在300微升盐水中，以形成储备溶液。测量其放射性后，将储备溶液在盐水中稀释，以获得500微升中0.037兆贝克勒尔的溶液。建立冷参比化合物的标定曲线，并解析线性回归方程：Y等于AX加B，得到A和B系数。X表示冷化合物的质量，Y表示曲线下的面积。检查放射性配体曲线下的面积，根据校准曲线和放射性配体曲线下的面积测量450纳米处的紫外吸光度。估计质量并测量放射性配体的活性。确保比活性大于每微摩尔1， 000千兆贝克勒。单击“更新图像”按钮以更新动物位置。通过借助屏幕底部的三个图像滑动光标来设置扫描区域。在 60 分钟扫描（共 60 帧一分钟）上设置实验。重用最初设置的所有其他参数。注入500微升放射性示踪剂，然后用300微升无菌0.9%氯化钠冲洗管子。同时，单击“开始采集”以开始扫描。打开扫描重建软件，然后打开数据集，查找在扫描文件夹中创建的文件名参数文件。选择感兴趣的同位素。请注意，列表模式参数允许在此步骤中选择多个同位素。设置不同的重建参数，如体素大小、子集数和迭代，如手稿中所述。选择输出格式为NIFTI，然后选择开始SPECT重建。使用扁平的金属刮刀和剃须刀片来剖析大脑感兴趣的区域。将组织置于两毫升离心管中并称量获得的组织。将样品放入装有一毫升无钙和无镁HBSS的两毫升离心管中，然后在室温下以300倍G离心两分钟，并在不干扰沉淀的情况下除去上清液。加入1， 900微升酶混合物，并在37摄氏度下孵育15分钟，同时每五分钟倒置搅拌一次管。加入30微升酶混合物两个。用1， 000微升移液器来回轻轻搅拌30次。然后，将混合物在37摄氏度下孵育15分钟，同时每五分钟倒置搅拌一次管。用200微升移液管和10微升移液器轻轻来回混合，然后将混合物在37摄氏度下孵育10分钟以解离组织。用70微米的细胞过滤器过滤细胞，并加入10毫升无钙和无镁的HBSS。在室温下以300倍G离心10分钟，并在不干扰沉淀的情况下除去上清液。对于髓鞘耗竭，用400微升髓鞘去除缓冲液重悬沉淀，然后加入100微升髓鞘去除珠，然后在4摄氏度下孵育15分钟。加入5毫升髓鞘去除缓冲液，室温下离心300倍G，持续10分钟。加入500微升髓鞘去除缓冲液，并将悬浮的沉淀放入磁场柱中。用一毫升髓鞘去除缓冲液洗涤柱四次。在室温下以300倍G离心两分钟，并在不干扰沉淀的情况下除去上清液。短暂涡旋以解离细胞并加入五微升的Fc Block CD32。加入100微升感兴趣的一抗混合物，并在4摄氏度下孵育20分钟。在4摄氏度下以350倍G离心5分钟，并在不干扰沉淀的情况下除去上清液。将试管倒置在软纸上。短暂涡旋试管，然后加入100微升二抗混合物，并在4摄氏度下孵育15分钟。加入一毫升髓鞘去除缓冲液，重复离心。丢弃上清液，在软纸上倒置印管。将细胞重悬于250微升无菌PBS中，并直接进行细胞分选。野生型大鼠的体内SPECT扫描显示，脂多糖注射部位的CLINDE结合高于大脑的对侧区域。接受荧光活化细胞分选放射性配体处理组织或FACS-RTT的离体样品显示仅在小胶质细胞中存在较多的CLINDE结合位点，表明大脑同侧CLINDE信号的细胞起源是小胶质细胞。TgF344-AD大鼠12个月时易位蛋白或TSPO结合的增加仅限于星形胶质细胞。在24个月大的大鼠中，由于星形细胞和小胶质细胞的改变，观察到TSPO结合的增加。在较老的TgF344-AD大鼠中，纹状体星形胶质细胞与野生型相比显示出降低的5HT2AR密度。当对人AD死后样本进行FACS-RTT时，与年龄匹配的对照相比，在AD受试者的星形胶质细胞和小胶质细胞中均观察到TSPO的皮质过表达。使用放射性时，请遵循无线电防护说明，并穿戴适当的个人防护设备。然后，在手术后，确保对工作环境进行净化。在此程序之后，可以在分离的细胞群上轻松进行蛋白质定量和基因表达分析。

Research

JoVE Journal

Neuroscience

Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue (FACS-RTT) to Determine the Cellular Origin of Radioactive Signal

An Optimized Procedure for Fluorescence-activated Cell Sorting (FACS) Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice

An Optimized Procedure for Fluorescence-activated Cell Sorting FACS Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice

荧光激活细胞分选（FACS）自主神经祖细胞的分离，从胎鼠的内脏器官的优化程序

During development neural crest (NC)-derived neuronal progenitors migrate away from the neural tube to form autonomic ganglia in visceral organs like the ...

科研

神经科学

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

使用胱天蛋白酶复分析，以确定细胞凋亡的下丘脑细胞模型

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

在小鼠大脑的各个区域端粒酶活性：非放射性端粒酶重复序列扩增法（TRAP）分析

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and ...

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

一<em&gt;在体外</em&gt;模型细胞病理生理学的球细胞脑​​白质营养不良的研究

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

在大鼠脑组织用荧光活化细胞分选，以检查细胞类型特异性基因表达

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

Fluorescence Activated Cell Sorting FACS and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

荧光活化细胞分选（FACS）和新鲜和冷冻大鼠脑组织的Fos表达神经元基因表达分析

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

在子宫内电途径研究神经元亚群的兴奋性和单细胞连接

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

温度梯度法测定果蝇幼虫的热偏好

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...

Primary Cell Culture of Purified GABAergic or Glutamatergic Neurons Established through Fluorescence-activated Cell Sorting

荧光活化细胞分选建立纯化的 Gabaep 或谷胱甘肽神经元的原代细胞培养

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can ...