Name: a rapid, simple, and standardized homogenization method to prepare antigen/adjuvant emulsions for inducing experimental autoimmune encephalomyelitis
Uploaded: 2022-12-09T14:00:00
Description: Watch this Scientific Journal Video about A Rapid, Simple, and Standardized Homogenization Method to Prepare Antigen/Adjuvant Emulsions for Inducing Experimental Autoimmune Encephalomyelitis at JoVE.c

作者要感谢隆德大学的动物饲养单位Camilla Björklöv和Agnieszka Czopek的支持，以及英国牛津大学肯尼迪风湿病研究所的Richard Williams的建设性批评和语言支持，以撰写这份手稿。

所有动物程序均按照瑞典动物研究委员会的惯例进行，并得到瑞典隆德-马尔默动物伦理委员会的批准（许可证号：M126-16）。
注意：该方法的示意图如图 1所示。
1. 材料准备
注意：将所有试剂无菌准备在无菌罩中，并等分并储存在指定的温度下。试剂可以储存长达 2 年而不会失去其效果。
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油包水乳剂，如抗原/弗氏佐剂，已被用于诱导EAE17超过半个世纪。目前还没有独立于人类影响的抗原乳剂制备标准化方法。使用注射器手动混合是大多数实验室的标准，但是这种方法非常耗时，通常会导致材料过度损失，并且质量因制备它的科学家而异。
本手稿中介绍的方法使用标准振荡均质机制备抗原/CFA 乳剂。此外，这里使用的所有试剂都可以等分并长...

免疫耐受性的破坏可导致自身免疫性疾病的产生，例如多发性硬化症（MS）。据估计，全世界有280万人患有MS1。尽管MS的确切原因在很大程度上仍未知，但自身反应性T细胞和B细胞的失调以及Treg功能的缺陷在该疾病的发病机制中起着重要作用2，3。
自身免疫性疾病的动物模型是研究潜在治疗方式的重要工具。实验性自身免疫性脑脊髓炎（EAE）模型已被对MS4感兴趣的研究人员使用了近一个世纪。在早期的实验中，该疾病的发病率相对较低。引入含有 分枝杆菌 和百日咳毒素的完全弗氏佐剂（CFA）能够在小鼠中一致地诱导EAE4。最重要的是，有必要将CFA与中枢神经系统（CNS）特异性抗原混合，以产生均匀的油包水乳液以诱导EAE。目前最常见的EAE模型是基于用致脑肽对小鼠进行主动免疫。小鼠的遗传背景在疾病易感性中起重要作用，髓鞘少突胶质细胞糖蛋白（MOG35-55）和髓鞘蛋白脂蛋白（PLP139-151）肽分别用于诱导C57BL / 6J和SJL小鼠的EAE，5。然而，也可以使用其他小鼠品系和CNS衍生的肽。
CFA/肽乳剂的质量是决定主动免疫EAE模型6中疾病外显率的关键因素。必须通过将溶解在水性缓冲液中的致脑肽与CFA混合来制备均匀的油包水乳液，否则动物不会患上这种疾病。关于CFA/肽乳液的制备已经发表了许多方案。实例包括使用涡旋7、超声处理8、注射器和三通T连接器9、或仅使用一个注射器5。然而，所有这些方法都难以标准化，并且通常与冗长而复杂的协议相关联。
与上述所有方法相比，此处描述的用于乳液制备的简单方法具有人与人之间没有差异且相对较快的优点。乳液由均质机以设定的速度、时间和温度摇动试剂产生，确保快速一致的结果。除了在EAE模型中诱导疾病外，该方法还可用于研究其他自身免疫性疾病模型，例如胶原蛋白诱导的关节炎（CIA）和抗原诱导的关节炎（AIA）6。因此，预计该方法可用于在依赖于含有自身抗原的油包水乳剂的其他动物模型中持续诱导疾病，例如实验性自身免疫性神经炎（EAN）10，实验性自身免疫性甲状腺炎（EAT）11，自身免疫性葡萄膜炎（EAU）12和重症肌无力（MG）13。该方法还持续诱导一般免疫反应，例如延迟型超敏反应（DTH）6，因此可用于提供癌症和疟疾疫苗（见讨论）。
因此，已经开发了一种快速（总制备时间~30分钟），简单（所有试剂都可以提前制备并储存）和标准化（使用振荡均质器完成乳液）的方法，并在此介绍。使用该协议制备的CFA /抗原乳剂在自身免疫动物模型中始终诱导疾病。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL Injection syringe</td>
			<td>B. Braun</td>
			<td>9166017V&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Injection syringe</td>
			<td>Sigma-Aldrich</td>
			<td>Z683531</td>
			<td></td>
		</tr>
		<tr>
			<td>7 ml empty tubes with caps</td>
			<td>Bertin-Instruments</td>
			<td>P000944LYSK0A.0</td>
			<td>7 mL tube</td>
		</tr>
		<tr>
			<td>50 mL sterile centrifuge tube&#160;</td>
			<td>Fisher Scientific</td>
			<td>10788561</td>
			<td>50 mL tube</td>
		</tr>
		<tr>
			<td>Bordetella pertussis toxin</td>
			<td>Sigma-Aldrich</td>
			<td>P2980</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Dispersant, light mineral oil</td>
			<td>Sigma-Aldrich</td>
			<td>M8410</td>
			<td>Store at RT</td>
		</tr>
		<tr>
			<td>Emulsion kit</td>
			<td>Bertin-Instruments</td>
			<td>D34200.10 ea</td>
			<td>Containing a tube, cap, and plunger</td>
		</tr>
		<tr>
			<td>Incomplete Freund&#39;s Adjuvant</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;F5506</td>
			<td>Store at +4 &#176;C</td>
		</tr>
		<tr>
			<td>Mycobacterium tuberculosis, H37RA</td>
			<td>Fisher Scientific</td>
			<td>DF3114-33-8</td>
			<td>Store at +4 &#176;C</td>
		</tr>
		<tr>
			<td>Mastersizer 2000&#160;</td>
			<td>Malvern Panalytical</td>
			<td>N/A</td>
			<td>Particle size analyzer</td>
		</tr>
		<tr>
			<td>Minilys-Personal homogenizer</td>
			<td>Bertin-Instruments</td>
			<td>P000673-MLYS0-A</td>
			<td>Shaking homogenizer</td>
		</tr>
		<tr>
			<td>MOG 35-55 Peptide&#160;&#160;&#160;</td>
			<td>Innovagen</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Montanide ISA 51 VG</td>
			<td>Seppic</td>
			<td>36362Z</td>
			<td>FDA-approved oil adjuvant</td>
		</tr>
		<tr>
			<td>Pall Acrodisc Syringe Filters 0.2 &#956;m</td>
			<td>Fisher Scientific</td>
			<td>17124381</td>
			<td>Sterlie filter</td>
		</tr>
		<tr>
			<td>PBS, Ca2+/Mg2+ free</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190144</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>Phase-Constrast Microscope</td>
			<td>Olympus</td>
			<td>BX40-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Steel Beads 3.2 mm</td>
			<td>Fisher Scientific</td>
			<td>NC0445832</td>
			<td>Autoclave and store at RT</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>648463</td>
			<td>Store at RT</td>
		</tr>
	</tbody>
</table>

a rapid, simple, and standardized homogenization method to prepare antigen/adjuvant emulsions for inducing experimental autoimmune encephalomyelitis

实验性自身免疫性脑脊髓炎（EAE）与多发性硬化症（MS）具有相似的免疫学和临床特征，因此被广泛用作识别新药物靶点以更好地治疗患者的模型。MS 的特征是几种不同的病程：复发缓解型 MS （RRMS）、原发性进行性 MS （PPMS）、继发性进行性 MS （SPMS） 和罕见的进行性复发型 MS （PRMS）。虽然动物模型不能准确地模仿所有这些对比鲜明的人类疾病表型，但有一些EAE模型反映了MS的一些不同临床表现。例如，C57BL / 6J小鼠中髓磷脂少突胶质细胞糖蛋白（MOG）诱导的EAE模仿人PPMS，而SJL / J小鼠中髓磷脂蛋白（PLP）诱导的EAE类似于RRMS。其他自身抗原，如髓鞘碱性蛋白（MBP）和许多不同的小鼠品系也用于研究EAE。为了在这些自身抗原免疫EAE模型中诱导疾病，制备油包水乳剂并皮下注射。大多数EAE模型还需要注射百日咳毒素才能发展疾病。为了获得一致且可重现的EAE诱导，需要详细的方案来制备试剂以产生抗原/佐剂乳剂。这里描述的方法利用标准化方法来生成油包水乳液。它简单快速，使用振荡均质机代替注射器来制备质量控制乳液。

用于制备CFA/MOG乳液的快速、简单和标准化方案如图 1所示。这种方法最近在别处有描述6.CFA / MOG乳液也可以用其他方法制备，例如传统的注射器方法或通过涡旋法制备。这里通过评估乳液的质量来比较这些方法。所有方法都生产油包水乳液;通过在载玻片上加入一小滴，然后在相差显微镜下观察来评估这些乳液的均匀性和质量。通过振荡均质法生产的乳液显?...

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A Rapid, Simple, and Standardized Homogenization Method to Prepare Antigen/Adjuvant Emulsions for Inducing Experimental Autoimmune Encephalomyelitis

为了诱导实验性自身免疫性脑脊髓炎，一种多发性硬化症的动物模型，用含有自身抗原的油包水乳剂对小鼠进行免疫并完成弗氏佐剂。虽然存在用于制备这些乳液的几种方案，但本文介绍了一种用于乳液制备的快速、简单和标准化的均质化方案。

一种快速、简单、标准化的均质化制备抗原/辅助乳剂以诱导实验性自身免疫性脑脊髓炎的方法

Experimental autoimmune encephalomyelitis (EAE) shares similar immunological and clinical features with multiple sclerosis (MS), and is therefore widely ...

实验性自身免疫性脑脊髓炎或EAE是一种动物模型，用于为多发性硬化症患者开发新的和改进的治疗方法。EAE中诱发疾病需要完美的油和水乳液才能获得一致的结果。这里介绍的乳液制备方法不受人为影响，从而限制了从一个实验到另一个实验的不一致。尽管该方法已针对EAE进行了优化，但它也可用于其他自身免疫性疾病模型以及测试癌症疫苗。在100毫克冻干结核分枝杆菌H37 RA和5个3.2毫米钢珠中制备CFA浆料到7毫升管中。以最高速度设置在均质机中摇动试管60秒。向试管中加入五毫升IFA佐剂，并以最高速度再次摇晃60秒。然后用移液器将浆液转移到新管中，留下珠子并储存在四摄氏度直至使用。要制备CFA/肽乳液，请下载补充文件一以计算每种试剂所需的量。然后将螺旋盖管和要使用的试剂放在冰上。接下来，添加乳液组分。首先是PBS，然后是肽，CFA浆液。最后，IFA。用盖子牢固地关闭管子，将其拧紧并松开几次。用手剧烈摇动试管 5 到 10 秒以预混合试剂。将管子放入振荡均质器中，并用杆固定。将速度设置为最高设置，将时间设置为 60 秒。运行完成后，将试管放在冰上三分钟，然后使用相同的设置再重复运行一两次。从一分钟开始以 300 G 离心管以去除滞留的空气并压实乳液。从管子上取下盖子。将柱塞插入管中，然后缓慢向下推，直到到达乳液的顶部。通过扭转管子底部的卡扣外壳将其卸下。接下来，从所有注射器中取出柱塞，并在注射器中加入针头。然后将注射器的背面连接到管底部的专用锁上，并用短促的扭转将其锁定。通过轻轻推动柱塞将乳液从管转移到注射器中。当乳液达到注射器的0.15毫升刻度时停止。将注射器与管子分开，并小心地插入柱塞，注意没有空气进入该注射器。推动柱塞，直到乳液从针头中出来，并对管中存在的其余乳液重复该过程。为了分析水和油颗粒的大小，将一小滴乳液添加到显微镜载玻片中，用盖玻片将其涂抹掉，然后以圆周运动用力推动以压平乳液。在放大倍数为400倍的相差显微镜下检查涂抹的乳液，并聚焦在具有单层乳液的场上。确保小的、均匀的、灰色或白色的颗粒是可见的。通过均质方法生产的乳液显示出灰色或白色的豆形颗粒，代表微小的水滴和油乳液。相比之下，使用注射器方法产生的乳液液滴更大更宽。在涡旋法中，观察到与乳液中捕获的气泡相对应的大灰色或黑色颗粒。使用激光折射粒度分析仪测量的水和油乳液的粒度分布随时间的变化在六周内没有显示出颗粒大小的任何变化。新鲜的3周龄和15周龄的乳剂在整个实验过程中测试的所有动物中都诱导了相似的EAE疾病。小鼠在所有实验中都出现了类似的疾病模式，表明这里介绍的均质化方法产生了以一致和可重复的方式诱导EAE的乳液。在为实验制作乳液之前，请下载补充文件编号一，以计算减少浪费所需的每种组分的量。这里介绍的方法不仅可用于临床前研究的乳剂制备标准化，而且可用于将来在临床上制备癌症疫苗。

Research

JoVE Journal

Immunology and Infection

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

鼠标垂体免疫原的制备诱导实验性自身免疫性垂体炎

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

科研

免疫与感染

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

SJL小鼠诱导实验性自身免疫性垂体炎

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

在实验性自身免疫性脑脊髓炎（EAE）抗性品系小鼠继转移和抗原挑战克服反应迟钝

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice

Myelin Oligodendrocyte Glycoprotein MOG35-55 Induced Experimental Autoimmune Encephalomyelitis EAE in C57BL/6 Mice

髓鞘少突胶质细胞糖蛋白（MOG<sub&gt; 35-55</sub&gt;）诱导实验性自身免疫性脑脊髓炎（EAE）的C57BL / 6小鼠

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While ...

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

在免疫细胞在不同组织与疾病相关的分布和小鼠实验评价性自身免疫性脑脊髓炎的诱导

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

一个简单的流式细胞仪法测量葡萄糖的摄取和葡萄糖转运表达单核细胞亚群的全血

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of ...

Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies

简单而高效的生产和鼠标髓鞘少突胶质糖蛋白的纯化的实验性自身免疫性脑脊髓炎研究

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses ...

Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo

体内自身免疫性脑髓炎实验性神经血管内障碍和胶质屏障的可视化

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

实验自身免疫性脑脊髓炎中枢神经系统淋巴细胞渗透的流量细胞计量分析

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...