The authors would like to acknowledge Dr. Ellen M. Fox for her work regarding this model of local anaphylaxis. This work was supported by the Uniformed Services University of the Health Sciences grant number R073UE.

1，小鼠敏感<ol><li>通过在1×PBS中稀释的OVA的20毫克/毫升的最终浓度制备OVA原液。冻结下在冷冻管并储存等分在-80℃下以供将来使用。 </li><li>带来的20毫克/毫升OVA股票的一个或多个等分试样至室温。在1X PBS稀释的OVA为1毫克/毫升的最终浓度在5ml的聚苯乙烯圆底管帽。轻轻振荡混匀。 </li><li> 1的比例：在保持含有1毫克/毫升OVA上的旋涡设置在中等速度的聚苯乙烯管中，同时继续涡管，以产生1加彻底匀浆Imject明矾（40毫克/毫升）逐滴加入等体积明矾免疫原。 </li><li>将瓶盖上的管带管的涡流。转涡旋上最低的设置，并允许OVA吸附到明矾30分钟。 <ol><li>不要让大量的时间来准备OVA /明矾和致敏小鼠之间传递。如果增感，不能直接OVA /明矾preparat后完成离子，商店OVA /明矾在室温下。旋涡使用前1分钟。 </li></ol></li><li>装入1 ml胰岛素注射器以OVA /明矾的混合物。顶盖采用27号针头的注射器。 </li><li>注入100μl的OVA /明矾到BALB / c小鼠的腹膜为每只小鼠的50微克的OVA和2mg明矾剂量。 </li><li>为阴性对照，致敏另一组小鼠以PBS中。制备1：明矾和PBS的混合物中同样地在上面列出，省略OVA。 </li><li>敏感的所有小鼠（OVA和PBS对照），每周一次，连续3周，共注射3次。最后一次注射后，请等待至少2周进行局部过敏反应试验之前。 </li></ol> 2，局部过敏反应含量<ol><li>带来的OVA原液至室温。在1.5ml微量离心管中，稀释20毫克/毫升OVA库存到5毫克/毫升，用PBS的终浓度（1:4稀释的OVA在1X PBS）。轻轻振荡混匀。 </li><li>麻醉采用RC 2 &lt;老鼠/ SUP&gt;啮齿动物麻醉系统（或类似的汽化输送系统）。填写水库异氟醚，然后打开氧气气流。小鼠放置在感应腔和异氟醚调节控制转盘至5％，开始麻醉。一旦老鼠被完全麻醉，由于缺乏流动性的指示，调整异氟烷控制转盘至3％，以维持深麻醉。麻醉在此过程的目的是为了固定所述动物。耳和尾静脉注射不会引起显著疼痛或痛苦的动物。 注：异氟醚可以让人类的生殖系统产生负面影响。请务必在通风良好的室内使用，并附上清除罐到感应室中和废气。如果您在使用异氟醚是孕妇，戴上碳滤光罩，以尽量减少风险。 </li><li>负载10微升5毫克/毫升的OVA到3月10日CC胰岛素注射器上铺上一个31 G针。加载另一个3/10毫升的胰岛素注射器，10微升的1X PBS。 </li><li>用解剖显微镜，注入10微升的OVA到麻醉小鼠的右耳为50微克OVA的挑战剂量。其他的剂量，例如20微克，也是可接受的。 <ol><li>为了稳定耳朵注射，包装胶带（粘性的一面朝上），围绕一个15毫升的锥形管。右耳的背侧面固定到磁带上。将针头斜面朝上两叶耳之间。慢慢地注入卵于耳组织的中心，注意不要打任何血管。 </li></ol></li><li>用上述同样的方法，注入10微升PBS中进左耳作为阴性对照。 </li><li>等待3分钟。 <ol><li>在此期间，将鼠标的抑止住在热灯或温水​​浴1分钟，扩张血管的尾巴。 </li></ol></li><li>用1毫升注射器胰岛素盖有27G的针头，慢慢注入200微升0.5％伊文思蓝染料到尾vEIN鼠标。快速注入整个加入200μl之前可能破坏血管结构被施用。如果出现这种情况，试图对尾巴的相对侧染料的剩余体积注入静脉。 </li><li>等待10分钟。 </li><li>安乐死鼠标和删除使用镊子和手术剪两个耳朵。 <ol><li>抓耳的边缘与镊子，使得没有任何压力被施加到注射部位处的耳的中心。去除尽可能多的耳组织尽可能通过切割接近，但不通过，软骨存在于耳朵的底部的脊。少量的皮毛上可能存在的切除耳;这并不会影响该测定。 </li></ol></li><li>分装700微升甲酰胺用1.5 ml离心管中。准备每穗一管（每只小鼠两管）。 注意：甲酰胺是致畸，致突变和刺激性。戴上手套，保护眼睛和面部防护。搬运时，孕妇应谨慎使用Š甲酰胺可能是胎儿中毒。 </li><li>添加耳甲酰胺，盖上盖子，并在干热浴中63℃孵育过夜。耳朵可能仍然会出现孵育后略带蓝色;然而大多数的染料将被提取到甲酰胺。 </li><li>加入300μl各个样品，一式两份，向96孔板中。避免转印皮毛，如果它是存在于样品中。确保没有气泡存在于所述孔中，因为它们会影响到吸光率读数。 </li><li>加入300μl甲酰胺中，一式两份，向96孔板中。这些孔将被用作空白。 </li><li>用分光光度计在620nm处测量吸光度。减甲酰胺井从每个样品的读数。 注：根据批准的统一服务大学实验动物管理和使用委员会的协议进行所有的实验。 </li></ol>

<ol>
	<li>Kojima, T., 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=Mast+cells+and+Bbasophils+are+selectively+activated+in+vitro+and+in+vivo+through+CD200R3+in+an+IgE-independent+manner.">Mast cells and Bbasophils are selectively activated in vitro and in vivo through CD200R3 in an IgE-independent manner.</a> The Journal of Immunology. 179 (10), 7093-7100 (2007).</li><li>Parikh, S. A., Cho, S. H., Oh, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preformed+enzymes+in+mast+cell+granules+and+their+potential+role+in+allergic+rhinitis.">Preformed enzymes in mast cell granules and their potential role in allergic rhinitis.</a> Current Allergy and Asthma Reports. 3 (3), 266-272 (2003).</li><li>Amin, K. <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+mast+cells+in+allergic+inflammation.">The role of mast cells in allergic inflammation.</a> Respiratory Medicine. 106 (1), 9-14 (2012).</li><li>Nakaya, M., Takeuchi, N., Kondo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+localization+of+histamine+receptor+subtypes+in+human+inferior+turbinates.">Immunohistochemical localization of histamine receptor subtypes in human inferior turbinates.</a> Annals of Otology, Rhinology &amp; Laryngology. 113 (7), 552-557 (2004).</li><li>Ramsdell, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+Trypan+Blue+to+demonstrate+the+immediate+skin+reaction+in+rabbits+and+guinea+pigs.">The use of Trypan Blue to demonstrate the immediate skin reaction in rabbits and guinea pigs.</a> The Journal of Immunology. 15 (4), 305-311 (1928).</li><li>Chase, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+sensitization+of+animals+with+simple+chemical+compounds:+X.+Antibodies+inducing+immediate-type+skin+reactions.">Studies on the sensitization of animals with simple chemical compounds: X. Antibodies inducing immediate-type skin reactions.</a> The Journal of Experimental Medicine. 86 (6), 489-514 (1947).</li><li>Goose, J., Blair, A. M. J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+cutaneous+anaphylaxis+in+the+rat,+induced+with+two+homologous+reagin-like+antibodies+and+its+specific+inhibition+with+disodium+cromoglycate.">Passive cutaneous anaphylaxis in the rat, induced with two homologous reagin-like antibodies and its specific inhibition with disodium cromoglycate.</a> Immunology. 16, 749-760 (1969).</li><li>Ovary, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+cutaneous+anaphylaxis+in+the+mouse.">Passive cutaneous anaphylaxis in the mouse.</a> The Journal of Immunology. 81 (4), 355-357 (1958).</li><li>Ovary, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immediate+reactions+in+the+skin+of+experimental+animals+provoked+by+antibody-antigen+interactoin.">Immediate reactions in the skin of experimental animals provoked by antibody-antigen interactoin.</a> Progress in Allergy. 5, 459-508 (1958).</li><li>Akiyama, H., 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=Quantitiative+evaluation+of+passive+cutaneous+anaphylaxis+(PCA)+using+a+hand-held+spectrophotometer.">Quantitiative evaluation of passive cutaneous anaphylaxis (PCA) using a hand-held spectrophotometer.</a> Biological &amp; Pharmaceutical Bulletin. 19 (8), 1112-1114 (1996).</li><li>Teshima, R., 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=Simple+spectrophotometric+analysis+of+passive+and+active+ear+cutaneous+anaphylaxis+in+the+mouse.">Simple spectrophotometric analysis of passive and active ear cutaneous anaphylaxis in the mouse.</a> Toxicology Letters. 95 (2), 109-115 (1998).</li></ol>

The authors have nothing to disclose.

许多过敏性疾病的标志物被用于评估下面的变应原激发过敏性反应，包括在气道激发的设置改变循环组胺，Th2型细胞因子的产生和细胞招募的水平进入支气管肺泡灌洗液的强度。而在这些参数监测的变化是研究过敏反应重要的是，生物标记物不总是关联与临床过敏性疾病。在本协议中所描述的地方过敏反应试验提供了本地化的血管通透性为局部过敏性疾病的一个相关可重复的测量。用蓝色染料外?...

I型超敏反应是由IgE介导的肥大细胞和嗜碱性粒细胞的表面上的抗原诱导的交联介导的。这导致细胞脱颗粒和血管活性和炎症介质如组胺，类胰蛋白酶，和血小板活化因子2的释放。以下预成形介体的脱颗粒过程中释放细胞，肥大细胞合成和释放前列腺素和白三烯，这进一步增加血管渗透性，3。最初的临床反应迅速发生，并且被称为“直接反应”。在皮肤中，风团和耀斑响应的抗原激发分钟内很容易地看到。根据这一挑战的剂量，就可以看到一个“晚期反应”几个小时后。后期肿胀是由 ​​于局部水肿和白细胞募集到组织2。组胺，一般被认为是主要的介体参与立即过敏性反应，作用于组胺受体1（HR1）表示对血管和组胺受体2（HR2）表达平滑肌。的联合作用增加了血流量和血管通透性在炎症4的位点。 多种过敏的动物模型已经发展为了研究参与过敏性炎症的机制，包括过敏性哮喘，全身性过敏反应，及本地过敏反应模型。静脉给药染料被用来衡量在动物模型中的局部过敏反应近一个世纪以来，随着出版物描述了这一技术可以追溯到20世纪20年代5。兔和豚鼠分别用于测试速发型超敏反应的第一个动物模型，以及最敏感的反应普遍发现在耳朵5,6。该法后来被证实为7大鼠和小鼠8使用。 从历史上看，各种实验方法已被使用，包括在注射之前，染料，染料注射之前注射抗原的注射抗原，以及同时注射的染料和抗原。静脉内给予染料作为测定过敏性反应的装置是一种多用途的测定法，它可以用于测量有源，无源，和反向被动反应的5,9。许多染料已被利用来评估过敏性反应，包括锥虫蓝，滂胺天蓝，伊文思蓝，嘉基蓝536，和印度油墨5,6,9。 0.5％伊文思蓝溶液是目前用于测定过敏性反应在皮肤上的标准染料。 挑战过敏性反应是短暂的;在10达到最大强度-染料注射15分钟，无反应是可见的，如果染料被施用多于30分钟挑战后，无论使用9的动物物种。染料外渗的定量原创LY获得通过测量风团大小的蓝色染料7-9所示。此外，脱颗粒的肥大细胞的数量可以通过从反应的部位切除的皮肤组织和甲苯胺蓝7染色来定量。肥大细胞脱颗粒经常被用来作为标记物用于皮肤，IgE介导的过敏性反应，如肥大细胞是表达高亲和力IgE受体了FcεRI主本地细胞群。分光光度技术测定染料外渗进入组织的被动皮肤过敏反应（PCA）在大鼠10和鼠标11，在20世纪90年代开发的。 以下本地过敏试验方案是改编自小岛等 。1，并利用鸡蛋卵白蛋白（OVA）作为抗原诱发过敏性反应。然而，可以使用的抗原相比OVA其他如果需要的话。该测定，然后使用伊文思蓝染料以监测变化的血管permeabilit发生由于肥大细胞的IgE交联和组胺释放年。

<table border="0" cellpadding="0" cellspacing="0" width="796">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Name of Material/ Equipment</td>
			<td style="width:167px;">Company</td>
			<td style="width:135px;">Catalog Number</td>
			<td style="width:225px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">BALB/c Mice</td>
			<td style="width:167px;">The Jackson Laboratory</td>
			<td style="width:135px;">651</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">PBS pH 7.4</td>
			<td style="width:167px;">Quality Biologic</td>
			<td style="width:135px;">114-058-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Ovalbumin</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:135px;">A5503-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Imject Alum</td>
			<td style="width:167px;">Thermo Scientific</td>
			<td style="width:135px;">77161</td>
			<td style="width:225px;">Mix thoroughly before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Evans Blue Dye</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:135px;">E2129</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Formamide</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:135px;">295876</td>
			<td style="width:225px;">99%+ Spectrophotometric grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Isoflurane, USP</td>
			<td style="width:167px;">Phoenix</td>
			<td style="width:135px;">NDC 57319-474-06</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">1cc Insulin syringes</td>
			<td style="width:167px;">BD</td>
			<td style="width:135px;">329654</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">3/10 cc Insulin syringe with 31G needle</td>
			<td style="width:167px;">Terumo</td>
			<td style="width:135px;">NDC 100861</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">27G Needles</td>
			<td style="width:167px;">BD</td>
			<td style="width:135px;">305109</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Forceps</td>
			<td style="width:167px;">F.S.T.</td>
			<td style="width:135px;">11000-12</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Surgical scissors</td>
			<td style="width:167px;">F.S.T.</td>
			<td style="width:135px;">14070-12</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">5ml Polystyrene round-bottom tubes</td>
			<td style="width:167px;">BD Falcon</td>
			<td style="width:135px;">352058</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">1.5ml Microcentrifuge tubes</td>
			<td style="width:167px;">Medical Supply Partners</td>
			<td style="width:135px;">15-1151</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">15ml Conical tubes</td>
			<td style="width:167px;">BD Falcon</td>
			<td style="width:135px;">352097</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Flat-bottom 96 well plate</td>
			<td style="width:167px;">Costar</td>
			<td style="width:135px;">3590</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Scotch tape</td>
			<td style="width:167px;"></td>
			<td style="width:135px;"></td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">RC2 Rodent Anesthesia System</td>
			<td style="width:167px;">VetEquip</td>
			<td style="width:135px;">922100</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Vortex Genie 2</td>
			<td style="width:167px;">Scientific Industries</td>
			<td style="width:135px;">SI-0236</td>
			<td style="width:225px;">Model G560 with 3 inch platform</td>
		</tr>
	</tbody>
</table>

measuring local anaphylaxis in mice

过敏性反应是肥大细胞和嗜碱性粒细胞，并随后释放血管活性和促炎介质的活化的结果。暴露于变应原的致敏个体可能会导致因不同轻微的红斑到危及生命的过敏反应临床症状。在实验室中，各种动物模型已经发展到理解驱动过敏性反应的机制。在这里，我们描述了用于测量变化，血管通透性量化局部过敏性反应的详细方法。局部过敏反应测定最早是在20世纪20年代的报道，并已改编自Kojima 等人发表的技术。在2007年1，在该试验中，敏化，以OVA小鼠左耳用车辆和在右耳用OVA激发。这后面是静脉注射伊文思蓝染料。注射伊文思蓝后十分钟，将动物安乐死，并在已渗出的染料到耳朵在一夜之间甲酰胺提取。所提取的染料的吸光率，然后定量用分光光度计。这种方法确实导致的局部过敏反应的可视化和可量化的表现。

已成功进行了试验动物都会有皮肤和眼睛呈现蓝色。 PBS致敏动物不应反应以PBS或OVA激发，因此两个耳朵应保持白色（ 图1A）。 OVA致敏的动物中，耳朵接收PBS挑战（左）应该是全白或在注射部位的局部的方式轻轻蓝色。耳接收OVA激发（右）应逐渐变暗的蓝色染料施用（ 图1B）之后的10分钟期间成为。 PBS致敏的动物将具有可忽略的吸光度读数为与PBS和OV...

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Measuring Local Anaphylaxis in Mice

过敏性反应，其特征在于由肥大细胞和嗜碱性粒细胞的活化，是由交联的IgE和炎症介质的释放驱动。过敏性反应的定量评估可以用伊文思蓝染料监察过敏原刺激后改变血管通透性来实现。

小鼠量度本地过敏反应

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

Research

JoVE Journal

Immunology and Infection

19.1K 次观看.  Uniformed Services University of the Health Sciences. 过敏性反应，其特征在于由肥大细胞和嗜碱性粒细胞的活化，是由交联的IgE和炎症介质的释放驱动。过敏性反应的定量评估可以用伊文思蓝染料监察过敏原刺激后改变血管通透性来实现。 

视频: Measuring Local Anaphylaxis in Mice

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

在固定和活细胞，流式细胞仪测量钙蛋白酶活性

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

科研

免疫与感染

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

测量细菌感染小鼠和免疫反应李斯特菌</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Measurement of &#947;HV68 Infection in Mice

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human &gamma;-HVs, such as EBV and KSHV, has severely hindered detailed pathogenic studies. Murine &gamma;-herpesvirus 68 (&gamma;HV68) shares extensive genetic and biological similarities with human &gamma;-HVs and is a natural pathogen of murid rodents2. As such, evaluation of &gamma;HV68 infection of mice inbred strains at different stages of viral infection provides an important model for understanding viral lifecycle and pathogenesis during &gamma;-HVs infection.
Upon intranasal inoculation, &gamma;HV68 infection results in acute viremia in the lung that is later resolved into a latent infection of splenocytes and other cells, which may be reactivated throughout the life of the host3,4. In this protocol, we will describe how to use the plaque assay to assess infectious virus titer in the lung homogenates on Vero cell monolayers at the early stage (5 - 7 days) of post-intranasal infection (dpi). While acute infection is largely cleared 2 - 3 weeks postinfection, a latent infection of &gamma;HV68 is established around 14 dpi and maintained later on in the spleen of the mice. Latent infection usually affects a very small population of cells in the infected tissues, whereby the virus stays dormant and shuts off most of its gene expression. Latently-infected splenocytes spontaneously reactivate virus upon explanting into tissue culture, which can be recapitulated by an infectious center (IC) assay to determine the viral latent load. To further estimate the amount of viral genome copies in the acutely and/or latently infected tissues, quantitative real-time PCR (qPCR) is used for its maximal sensitivity and accuracy. The combined analyses of the results of qPCR and plaque assay, and/or IC assay will reveal the spatiotemporal profiles of viral replication and infectivity in vivo.

Measurement of &amp;#947;HV68 Infection in Mice

&#947;-Herpesviruses (&#947;-HVs) establish life-long persistency in their host. Infection of mice with &#947;-HV68 provides a genetically tractable in vivo model for the characterization of the lifecycle/pathogenesis of &#947;HVs. This protocol describes the detection and quantitation of &#947;HV68 infection at acute and latent stages following infection by plaque-forming, infectious center, and qPCR assays.

γHV68感染小鼠的测量

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human ...

Intralymphatic Immunotherapy and Vaccination in Mice

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of vaccine to lymph nodes where antigen presenting cells can interact with lymphocytes for generation of the wanted immune responses. The strength and the type of immune responses induced also depend on the density or frequency of interactions as well as the microenvironment, especially the content of cytokines. As only a minute fraction of peripherally injected vaccines reaches the lymph nodes, vaccinations of mice and humans were performed by direct injection of vaccine into inguinal lymph nodes, i.e.&#160;intralymphatic injection. In man, the procedure is guided by ultrasound. In mice, a small (5-10 mm) incision is made in the inguinal region of anesthetized animals, the lymph node is localized and immobilized with forceps, and a volume of 10-20 &#956;l of the vaccine is injected under visual control. The incision is closed with a single stitch using surgical sutures. Mice were vaccinated with plasmid DNA, RNA, peptide, protein, particles, and bacteria as well as adjuvants, and strong improvement of immune responses against all type of vaccines was observed. The intralymphatic method of vaccination is especially appropriate in situations where conventional vaccination produces insufficient immunity or where the amount of available vaccine is limited.

Prophylactic and therapeutic vaccination often fails to stimulate strong immune responses due to week drainage of the vaccine to lymph nodes and consequently poor involvement of immune cells. By direct injection of vaccine to lymph nodes, so-called intralymphatic injection, vaccine efficacy can be strongly improved and vaccine doses can be reduced.

淋巴免疫治疗和疫苗接种小鼠

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of ...

Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of disease or occurrence of serious complications. Ektacytometry uses laser diffraction viscometry to measure the deformability of red blood cells subject to either increasing shear stress or an osmotic gradient at a constant value of applied shear stress. However, direct deformability measurements are difficult to interpret when measuring heterogenous blood that is characterized by the presence of both rigid and deformable red cells. This is due to the inability of rigid cells to properly align in response to shear stress and results in a distorted diffraction pattern marked by an exaggerated decrease in apparent deformability. Measurement of the degree of distortion provides an indicator of the heterogeneity of the erythrocytes in blood. In sickle cell anemia, this is correlated with the percentage of rigid cells, which reflects the hemoglobin concentration and hemoglobin composition of the erythrocytes. In addition to measuring deformability, osmotic gradient ektacytometry provides information about the osmotic fragility and hydration status of erythrocytes. These parameters also reflect the hemoglobin composition of red blood cells from sickle cell patients. Ektacytometry measures deformability in populations of red cells and does not, therefore, provide information on the deformability or mechanical properties of individual erythrocytes. Regardless, the goal of the techniques described herein is to provide a convenient and reliable method for measuring the deformability and cellular heterogeneity of blood. These techniques may be useful for monitoring temporal changes, as well as disease progression and response to therapeutic intervention in several disorders. Sickle cell anemia is one well-characterized example. Other potential disorders where measurements of red cell deformability and/or heterogeneity are of interest include blood storage, diabetes, Plasmodium infection, iron deficiency, and the hemolytic anemias due to membrane defects.

Here we present techniques to measure red cell deformability and cellular heterogeneity by ektacytometry. These techniques are applicable to general investigations of red cell deformability and specific investigations of blood diseases characterized by the presence of both rigid and deformable red cells in circulation, such as sickle cell anemia.

Ektacytometry 血中红细胞异质性的测定

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

酶联免疫吸附法表征小鼠胸腺依赖性和胸腺独立免疫球蛋白同种反应

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

Mouse Body Temperature Measurement Using Infrared Thermometer During Passive Systemic Anaphylaxis and Food Allergy Evaluation

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature readings is common, and they have been proven to be accurate and invaluable in this regard. However, this method of temperature measurement requires the mice to be anesthetized in order to insert the probe without injury to the animal. This limits the ability to observe other phenotypes of the mouse simultaneously. In order to investigate other phenotypes while measuring temperatures, rectal probes are not ideal, and another method is desired. Here, we introduce a noninvasive method of temperature measurement that foregoes the requirement for mouse anesthesia while maintaining equal reliability to rectal probes in measuring body temperature. We use an infrared thermometer that detects body surface temperatures at ranges between 2 and 150 mm. This method of body temperature measurement is successful in reliably replicating temperature change trends during passive system anaphylaxis experiments in mice. We show that body surface temperatures are about 2.0 &#176;C lower than rectal probe measurements, but the degree of temperature drop follows the same trend. Furthermore, we use the same technique to observe mice in a food allergy model to evaluate temperature and activity levels simultaneously.

Here we present a new method to accurately measure body temperature differences in passive systemic anaphylaxis (PSA) and food allergy mouse models using an infrared thermometer. This procedure has been accurately duplicated in previous PSA results.

红外测温仪在被动系统过敏反应和食品过敏评价中的应用

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature ...

Quantification of Atherosclerosis in Mice

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic inflammatory disease. Experimental atherosclerosis studies have elucidated the role of cholesterol and inflammation in the disease process. This has led to successful clinical trials with pharmaceutical agents that reduce clinical manifestations of atherosclerosis. Careful and well-controlled experiments in mouse models of the disease could further elucidate the pathogenesis of the disease, which is not fully understood. Standardized lesion analysis is important to reduce experimental variability and increase reproducibility. Determining lesion size in aortic root, aortic arch, and brachiocephalic artery are common endpoints in experimental atherosclerosis. This protocol provides a technical description for evaluation of atherosclerosis at all these sites in a single mouse. The protocol is particularly useful when material is limited, as is frequently the case when genetically modified animals are being characterized.

Murine models of atherosclerosis are useful tools to investigate pathogenic pathways on a molecular level, but require standardized quantification of lesion development. This protocol describes an optimized method to determine lesion size in the major arterial vessels including the aortic root, aortic arch, and brachiocephalic artery.

小鼠动脉粥样硬化的定量

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

葡萄糖-6-磷酸异构酶（G6PI）诱导RA小鼠中iNKT细胞的采用免疫治疗

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

利用流细胞测定人类白细胞测定阿斯珀吉鲁斯紫锥体烟曲的噬菌体

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...