蒂娜马丁和大卫奥尔布赖特提供的专家畜牧业衷心赞赏。这项研究得到了补助的T32 - AI - 07285（HN），美国国立卫生研究院R25 2GM064133（TCL），1R03 HD061671 - 01，R24 - AI - 059830 - 06由美国国立卫生研究院。

1。动物 非洲爪蟾祥煦gilli杂交LG - 6和LG - 15 isogenetic克隆是从我们的繁殖地在罗切斯特大学（http://www.urmc.rochester.edu/smd/mbi/xenopus/index.htm） 。 LG - 6和LG - 15的份额相同的杂合的MHC单倍型（A / C），但不同的次要组织相容性位点（H）。从这些克隆后代，是由雌核发育，女性所产生的二倍体卵子是由紫外线照射精子（无DNA的贡献后代）被激活。 使用手套兼。有些人喜欢不穿，因为它是更难处理的青蛙（滑），实际上它似乎使青蛙不舒服。 2。纯化的GP96 15 / 0肿瘤（对两种肿瘤和次要的H - AGS） GP96净化先前已经描述2，3。简单地说，GP96是50-70％硫酸铵盐析，由ConA琼脂糖凝胶层析和DEAE纯化。蛋白质约20-50微克，可每1毫升的肿瘤组织。该制剂的纯度是由SDS - PAGE电泳和银染。 3。从轻微的H - AG -不同的LG - 6青蛙腹腔白细胞（PLS）的启发和收获<ol><li>长出了25毫升的隔夜大肠杆菌大肠杆菌文化在50 mL锥形管在37℃用颤抖的彗星。 </li><li>第二天，加热煮沸1小时杀死细菌。 </li><li>离心杀害大肠杆菌热大肠杆菌为2,000 RPM（1500克）在4 ° C 15分钟</li><li>去除上清，重悬细菌沉淀在1 / 10的原始文化在APBS量（2.5毫升） 。此时细菌培养就可以使用了，必须在24小时内使用。 </li><li>注入腹腔（IP）200（2英寸青蛙）或300μL（3英寸青蛙）的热量杀死细菌每青蛙准备使用25号5 / 8针。 </li><li> 3天注射后PLS收获腹腔灌洗。 </li><li> PL收获之前，成年青蛙tricaine甲烷磺酸钠碳酸氢钠缓冲长达5分钟，MS - 222（TMS）在0.1％水溶液浸泡麻醉停止，直到所有的运动（持续时间的大小和年龄而定）。动物治疗后10-20分钟内醒来。 </li><li>用少量70％乙醇消毒青蛙腹部。 </li><li>注入5毫升（2英寸青蛙）或10毫升（3英寸青蛙），在室温下预热使用一个18针1个半针进入腹膜腔的无菌APBS。取出针，轻轻按摩一分钟，以保证注入的缓冲区，体腔液达到平衡的青蛙。 </li><li>没有收集腹腔液针背面滴水成一个干净的50 mL锥形管的注射器，使用一个新的18针1个半针。确保检索尽可能多的初始注入量。要小心，以避免在中部地区的腹部地区的血管。 </li><li> PLS是收获后在浅水的容器中放青蛙，直到它是清醒的，此时它可以被放置在笼子里。 </li></ol> 4。 LG - 6 PLS朴素LG - 6收件人脉冲和过继转移<ol><li> PLS一次洗净冷APBS和离心机在1000转（750克），在4 ° C 10分钟</li><li>去除上清，重悬在APBS PLS。 </li><li>在浓度为1微克每5 × 10 5 PLS GP96与GP96脉冲PLS。 </li><li>一旦添加适量的GP96的PLS，混合pipeting和冰上孵育1小时。 </li><li>在1000转10分钟在4 ° C或1分钟14000转离心细胞，以消除任何未绑定的GP96。 </li><li>洗净的薪酬水平调查，以确保没有残留的GP96左边用冷水APBS 3X。 </li><li>重悬浓度在5 × 10 5每300μL的PLS脉冲PLS 。 </li><li>过继转移PLS IP使用25计5 / 8针的注射。注入300μL的动物（5 × 10 5细胞）脉冲PLS 。 </li><li>青蛙必须要植皮或肿瘤的挑战实验持续至少3天前催芽。 </li></ol> 5。皮肤移植实验皮肤移植排斥反应是在4，5，6 爪以及建立检测。在非洲爪蟾 ，收件人拒绝接受捐助者在21日至22日在18-22天的皮肤显示1或2 MHC单体型不匹配° C。相比之下，LG - 6和LG - 15只有轻微的H - AGS不同的克隆之间的皮肤移植将被拒绝更慢（30天）。然而，这种拒绝是加快受助THAT有催芽对未成年人的H - AGS捐助由以前的皮肤移植或从捐赠者的免疫纯化与GP96。根本没有排斥反应发生时，捐助者和接受者是基因完全相同的（例如，克隆或完全近交系动物）。因此，这个简单的技术特征GP96引起体内的免疫反应是非常强大的。 <ol><li>麻醉捐助青蛙，如3.7中所述。 注：只有最小的预防措施，需要采取以来爪生产总无菌程序需要在皮肤上，省却了强有力的抗微生物肽（例如，magainin）。事实上，治疗青蛙与任何消毒剂对皮肤不利。此外， 非洲爪蟾没有任何可以转移到人类病原体。因此，手套的使用是可选的。然而，所有的夹层法器需要进行高压灭菌，所有的解决方案必须是无菌的。 </li><li>剪切和删除的小（20毫米× 5毫米）腹侧皮肤一块从一台LG - 15捐款使用剪刀的青蛙（由于存在的irridophore色素细胞，银色的腹部皮肤出现）。 </li><li>放置在一个Petridish APBS的皮肤和保持它在冰上。操纵的皮肤组织非常轻柔，避免控股钳之间。 </li><li>使用剃刀或剪刀个别移植切成5毫米× 5毫米件。保持在上冰APBS的碎片。 </li><li>此时捐助青蛙是放置在浅水的容器中，直到它是清醒的，然后它被置于含有抗生素（浓度为5毫克/升Penstrep）的水。青蛙保存在两天此时它是在正常的水返回水用抗生素。在皮肤被删除的网站的小伤口并不需要缝合和在一个星期内愈合。 </li><li>麻醉收件人LG - 6青蛙。 </li><li>收件人的皮肤上背（背部）的一个小切口插入5毫米× 5毫米的皮肤下移植与银色的方。要小心，不要引进皮肤下的大气泡，因为这可能会导致流离失所，甚至移植的损失。 </li><li>确保您注意移植的剪刀和镊子的标记，因为这些都不是移植排斥反应的得分开始算一次。 </li><li> 24小时后的叠加主机皮肤的一部分需要从嫁接。 </li><li>麻醉收件人LG - 6青蛙。处理如5.1中所述的动物。 </li><li>使用高压灭菌剪刀剪出一个窗口，让周围嫁接的嫁接可以自由显示。小心不要触摸捐助者的皮肤或诱发出血。让我们在5.5中所述的青蛙恢复。 </li><li>马上开始得分嫁接草图。皮肤移植排斥反应是由嫁接皮肤销毁irridophores％。 </li><li>移植需要进行检查，每2-3天，但不需要这个麻醉的动物。为了形象化的移植，青蛙必须在petridish放置在解剖显微镜下。 </li></ol> 6。全安装移植皮肤的免疫组化青蛙全挂载免疫组先前已描述6。简单地说，青蛙是麻醉，在显微镜下放在一个无菌纸预湿毛巾与青蛙的水（无菌准备工作区域也）。连同少量的周围宿主皮肤移植的皮肤，然后收获，同样是与抗体染色细胞染色流式细胞仪分析 6 。需要使用蒸压文书和无菌缓冲区。染色后的皮肤是放在载玻片和盖玻片轻轻按下。此时的皮肤可以使用不同的细胞，已经渗透到移植物的种群荧光显微镜可视化。手术后的青蛙是用抗生素，如5.5节所述，在水中保持。并返回正常用水。小伤口，皮肤被删除，在一周内痊愈。 7。血白细胞的表征<ol><li>从青蛙的血之前，需要准备拉火焰无菌巴斯德移液器的“玻璃针”。罚款移液器端削尖的体视显微镜下。然后连接到移液器吸塑料管。 所有仪器，如镊子和剪刀需要使用前必须高压灭菌。 </li><li>还准备增加50个单位，每1毫升的APBS肝素冰冷APBS和肝素溶液10毫升。置于冰上的解决方案。使用所有的解决方案必须是无菌的。 </li><li>麻醉青蛙，如3.7中所述，并遵循相同的动物如5.1中所述的程序处理。 。 </li><li>切割后足以上的皮肤暴露跗背静脉。 </li><li>巴斯德吸管填写APBS +肝素液1-2毫升。 </li><li> “玻璃针”插入静脉，慢慢用口吸气，开始收集血液。重要的是有“玻璃针”APBS +肝素的解决方案，因为这将防止血液凝血和堵塞针尖。 </li><li> 1至2毫升血液可从一个平均大小的青蛙。 </li><li>青蛙的腿的小切口并不需要缝合伤口愈合一周内。 </li><li>在10分钟1000转离心血液在4 ° C。 </li><li>去除上清，洗细胞10毫升冷APBS 1X。 </li><li>覆盖2毫升血液（1-5 × 10 6细胞）到1毫升Histopaque 1.077的聚蔗糖（Sigma公司），在室温下预热，以单独的血白细胞，红细胞。 </li><li>在室温下在1000 RPM没有刹车离心20分钟，然后收集白细胞乐队。 </li><li>洗净在1400转的细胞与APBS 2X离心10分钟，4 ° C至去除残留的聚蔗糖。 </li><li>此时细胞准备与流式细胞仪分析抗体染色，或在体外培养实验。 </li></ol> 8。肿瘤移植含量<ol><li>大约一个星期前出一批新的15 / 0肿瘤细胞，并确保这些细胞开始生长良好的实验解冻。在下一节和3更详细的培养基是简洁的描述。 </li><li>展开15 / 0肿瘤细胞。 </li><li>在实验的当天，细胞计数和台盼蓝拒确定细胞死亡。细胞死亡，应小于5％。洗在1000转冷APBS 1X细胞，并在4 ° C离心10分钟。 </li><li>在肿瘤的培养基重悬细胞密度5 × 10 5月 15日/ 0细胞每300μL。 </li><li>移植5 × 10 5细胞300μL每青蛙数量皮下背侧（背面）的动物的一侧使用25号5 / 8针的注射。 </li><li>肿瘤的生长，将开始在2-3周后肿瘤的挑战。必须指出最初的肿瘤外观和肿瘤体积需要记录每2-3天。用游标卡尺测量肿瘤体积（高X长X宽）。 </li><li>一旦肿瘤生长到3500毫米3或青蛙开始寻找昏昏欲睡，它需要被安乐死，以防止不适。 </li></ol> 9。试剂所需： <ul><li>两栖磷酸盐缓冲液（APBS）：6.6 g / L的氯化钠，1.15 g / L的钠2 HPO 4，0.2 g / L的KH 2 PO。 pH值至7.5使用10N氢氧化钠和过滤器消毒过滤到0.2微米。 </li><li> Tricaine甲烷磺酸（TMS，MS - 222）（红新月化学研究CAS＃886-86-2）。 </li><li>碳酸氢钠（尔科技的S - 233 - 500）。 </li><li> Histopaque - 1077（Sigma - Aldrich公司10771-100毫升） </li><li>肝素钠盐（Sigma - Aldrich公司的H3149 - 50KU） </li><li> 爪 15 / 0肿瘤的培养基[更多详情参见3]：1 L的10 mL胰岛素，10毫升的非必需氨基酸，10毫升的青霉素，链霉素10微克Iscove DMEM培养基（GIBCO Invitrogen公司11965） / mL的卡那霉素3毫升primatone（谢菲尔德产品事业部），β2-巯基乙醇1毫升，3.02克碳酸氢钠在水中（pH值7.0）。这种介质是通过添加30％的双蒸水稀释至两栖动物渗透压，并辅以5％小牛血清，20％， 从非洲爪蟾肾脏细胞株A6 superantant，和0.25％的正常爪蟾血清。 </li></ul> 10。代表性的成果<img src="/files/ftp_upload/2026/2026fig1.jpg" alt="图1" /> 图1。Stereomicroscopic分析皮肤移植排斥反应的移植后12天。 LG - 6克隆青蛙（一）MHC相同的LG - 6植皮，（无排斥反应）或（B）一个MHC不同的远交捐助者（80％抑制）。箭头显示银色iridophore标志着健康（非拒绝）嫁接组织的色素细胞。 （*）标记的镊子不因拒绝。 <img src="/files/ftp_upload/2026/2026fig2.jpg" alt="图2" /> 图2。 GP96有利于肿瘤抗原的交叉呈现在爪蟾 。脉冲为1小时上冰APBS（阴性对照），1微克一个大肠杆菌纯化重组GP96 LG - 15 PLS（5 × 10 5 ）从15 / 0肿瘤组织纯化大肠杆菌文化，或1或0.5微克的GP96。 3洗涤后，细胞过继转移到LG - 15的成年受助人（1 × 10 6 /个人） 。三天后，现场15 / 0肿瘤细胞（5 × 10 5）通过皮下注射移植。每条曲线代表肿瘤的生长在一个青蛙的动力学。天挑战，当肿瘤最早出现的监测，并定期用卡尺（长x宽x厚）被确定肿瘤的大小。

<ol>
	<li>Kobel, H. R., Pasquier, D. u., L, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperdiploid+species+hybrids+for+gene+mapping+in+Xenopus.">Hyperdiploid species hybrids for gene mapping in Xenopus.</a> Nature. 279, 157-158 (1979).</li><li>Robert, J., Menoret, A., Basu, S., Cohen, N., Srivastava, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+conservation+of+the+molecular+and+immunological+properties+of+the+chaperones+gp96+and+hsp70.">Phylogenetic conservation of the molecular and immunological properties of the chaperones gp96 and hsp70.</a> Eur J Immunol. 31, 186-195 (2001).</li><li>Robert, J., Gantress, J., Cohen, N., Maniero, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+as+an+experimental+model+for+studying+evolution+of+hsp--immune+system+interactions.">Xenopus as an experimental model for studying evolution of hsp--immune system interactions.</a> Methods. 32, 42-53 (2004).</li><li>Chardonnens, X., Pasquier, D. u., L, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+skin+allograft+tolerance+during+metamorphosis+of+the+toad+Xenopus+laevis:+a+possible+model+for+studying+generation+of+self+tolerance+to+histocompatibility+antigens.">Induction of skin allograft tolerance during metamorphosis of the toad Xenopus laevis: a possible model for studying generation of self tolerance to histocompatibility antigens.</a> Eur J Immunol. 3, 569-573 (1973).</li><li>Du Pasquier, L., Bernard, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+suppression+of+the+allogeneic+histocompatibility+reactions+during+the+metamorphosis+of+the+clawed+toad+Xenopus.">Active suppression of the allogeneic histocompatibility reactions during the metamorphosis of the clawed toad Xenopus.</a> Differentiation. 16, 1-7 (1980).</li><li>Ramanayake, T., Simon, D. A., Frelinger, J. G., Lord, E. M., Robert, J. <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+study+of+T-cell+responses+to+skin+alloantigens+in+Xenopus+using+a+novel+whole-mount+immunohistology+method.">In vivo study of T-cell responses to skin alloantigens in Xenopus using a novel whole-mount immunohistology method.</a> Transplantation. 83, 159-166 (2007).</li><li>Robert, J., Ramanayake, T., Maniero, G. D., Morales, H., Chida, A. S., S, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+conservation+of+glycoprotein+96+ability+to+interact+with+CD91+and+facilitate+antigen+cross-presentation.">Phylogenetic conservation of glycoprotein 96 ability to interact with CD91 and facilitate antigen cross-presentation.</a> J Immunol. 180, 3176-3182 (2008).</li><li>Robert, J., Gantress, J., Rau, L., Bell, A., Cohen, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minor+histocompatibility+antigen-specific+MHC-restricted+CD8+T+cell+responses+elicited+by+heat+shock+proteins.">Minor histocompatibility antigen-specific MHC-restricted CD8 T cell responses elicited by heat shock proteins.</a> J Immunol. 168, 1697-1703 (2002).</li><li>Morales, H., Robert, J. <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+and+in+vitro+techniques+for+comparative+study+of+antiviral+T-cell+responses+in+the+amphibian+Xenopus.">In vivo and in vitro techniques for comparative study of antiviral T-cell responses in the amphibian Xenopus.</a> Biol Proced Online. 10, 1-8 (2008).</li><li>Maniero, G. D., Robert, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+conservation+of+gp96-mediated+antigen-specific+cellular+immunity:+new+evidence+from+adoptive+cell+transfer+in+xenopus.">Phylogenetic conservation of gp96-mediated antigen-specific cellular immunity: new evidence from adoptive cell transfer in xenopus.</a> Transplantation. 78, 1415-1421 (2004).</li></ol>

两栖类爪蟾是一个独特的多功能非哺乳动物模型来研究的免疫力。已经取得了许多重要的研究工具，如MHC的定义克隆LG - 6和LG - 15以及不同的细胞系和单克隆抗体及其在生物医学和免疫学研究中的广泛使用。使用这些工具，我们已经建立了在体外和体内实验研究热休克蛋白GP96如的能力，调解有力银特定抗轻微的H - Ag和抗肿瘤的T细胞反应。这个模型系统，我们可以进一步探讨在吸效应?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Reagents needed:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Amphibian Phosphate Buffered Saline (APBS): </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NaCl, 1.15 g/L </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Na2HPO4, 0.2 g/L </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>KH2PO </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10N NaOH </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tricaine Methane Sulfonate (TMS, MS-222) </td>
<td>&nbsp;</td>
<td>Crescent Research Chemicals </td>
<td>CAS#886-86-2</td>
<td>&nbsp;</td>
<tr>
<td>Sodium bicarbonate </td>
<td>&nbsp;</td>
<td>Fisher Scientific </td>
<td>S-233-500</td>
<td>&nbsp;</td>
<tr>
<td>Histopaque-1077</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>10771</td>
<td>100ml</td>
</tr>
<tr>
<td>Heparin Sodium Salt </td>
<td>&nbsp;</td>
<td>Sigma-Aldrich </td>
<td>H3149-50KU</td>
<td>&nbsp;</td>
<tr>
<td>Culture medium for Xenopus 15/0 tumors [see 3 for more details]:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Iscove DMEM basal medium</td>
<td>&nbsp;</td>
<td>Gibco-Invitrogen </td>
<td>11965</td>
<td>&nbsp;</td>
<tr>
<td>Insulin</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Non-essential amino acids</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-streptomycin </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Kanamycin</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Primatone</td>
<td>&nbsp;</td>
<td>Sheffield Products Division</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>&beta;2-mercaptoethanol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NaHCO3 </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>30% double distilled water </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>5% featal bovine serum</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>20% superantant from a Xenopus kidney cell line A6 </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>0.25% of normal Xenopus serum</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Materials and Equipment:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>50 and 15 ml conical centrifuge tubes (sterile)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>25 gauge 5/8 Precision Glide sterile needles</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>18 gauge 1&frac12; Precision Glide sterile needles</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1 ml Tuberculin Slip Tip Syringe sterile</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10 ml Slip Tip Syringe sterile</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1.5 ml MicroCentrifuge tubes (sterile)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>60 X 15 mm Polystyrene Petri Dishes sterile</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Razor blades</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>25 X 75 mm X 1 mm Premium Microscope Slides </td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10 cm glass petri dishes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>9" Pasteur Pipetes Durex Borosilicate Glass Cotton Plugged Disposable</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Tygon tubing</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Two # 5 Swiss Jeweler&#x2019;s Forceps</td>
<td>&nbsp;</td>
<td>Miltex Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micro Dissecting Spring Scissors McPherson-Vannas straight cutting edge 6 mm</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Helios calipers </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>High intensity illuminator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Hemacytometer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Un-bunsen burner</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>37&deg;C shaking incubator</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

comparative in vivo study of gp96 adjuvanticity in the frog xenopus laevis

我们已经开发的两栖类爪蟾蟾一个独特的非哺乳动物模型来研究在一定的热休克蛋白（HSP的）的能力，如GP96，以方便陪护抗原交叉呈现，引起先天免疫和适应性的T细胞反应。爪蟾的皮 ​​肤移植排斥反应研究的GP96引出经典MHC Ia类（Ia类）的有限制的T细胞反应的能力提供了一个极好的平台。此外， 非洲爪蟾模型系统还提供了一个有吸引力的替代小鼠探索的GP96反应生成对肿瘤有下调他们的Ia类分子，从而逃避免疫监视的能力。最近，我们开发了一种使用腹腔白细胞抗原提呈细胞（APCs）在爪蟾克隆收养细胞转移实验，并表明，GP96可以首相CD8 + T细胞在体内对未成年人的组织相容性皮肤抗原， 以及对非洲爪蟾胸腺肿瘤15反应/ 0，这并不表示Ia类分子。我们在这里描述，涉及到执行这些包括腹腔白细胞的启发，脉冲和收养的转移，以及皮肤移植和肿瘤移植实验的实验方法。此外，我们还描述的收获外周血白细胞使用流式细胞仪和增殖实验皮肤排斥和抗肿瘤反应的效应人口的进一步鉴定和分离。

Watch this Scientific Journal Video about Comparative in vivo Study of gp96 Adjuvanticity in the Frog Xenopus laevis at JoVE.com

Comparative in vivo Study of gp96 Adjuvanticity in the Frog Xenopus laevis

青蛙<em&gt;非洲爪蟾</em&gt;提供了一个有吸引力的替代非哺乳动物模型探讨热休克蛋白等作为GP96的能力，促进抗原特异性CD8 T细胞反应。我们目前的方法来研究<em&gt;在体内</em&gt; GP96促进皮肤和肿瘤抗原的交叉呈现。

比较在体内 GP96佐剂研究中的青蛙非洲爪蟾</em

We have developed in the amphibian Xenopus laevis a unique non-mammalian model to study the ability of certain heat shock proteins (hsps) such as gp96 to ...

comparative-vivo-study-gp96-adjuvanticity-frog-xenopus

Research

JoVE Journal

Immunology and Infection

Comparative in vivo Study of gp96 Adjuvanticity in the Frog Xenopus laevis

10.5K 次观看.  University of Rochester. 青蛙非洲爪蟾提供了一个有吸引力的替代非哺乳动物模型探讨热休克蛋白等作为GP96的能力，促进抗原特异性CD8 T细胞反应。我们目前的方法来研究在体内 GP96促进皮肤和肿瘤抗原的交叉呈现。

视频: Comparative in vivo Study of gp96 Adjuvanticity in the Frog Xenopus laevis

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

在Live主机转利什曼原虫寄生虫在体内成像

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

科研

免疫与感染

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

白细胞的归位和迁移的非侵入性的成像在体内</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

培养小胶质细胞的新生和成年的中枢神经系统

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

在体外和体内模型来研究细菌粘附于血管壁下流动条件

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

人胎盘及蜕膜器官培养研究在母胎界面感染

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

辅助性T细胞作为一种遗传方法来研究逆转录病毒转导机制控制其分化和功能

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

黏膜衬里流体为体内上呼吸道免疫介质水平量化的无创性取样

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

人单核细胞趋化体外和小鼠淋巴细胞贩运体内

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

人类舱内单聚物研究病原体、共脉和宿主肠道上皮细胞之间的相互作用

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

活体细胞毒性试验研究肿瘤特异性 CD8+ t 细胞反应中的免疫调节

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...