作者要感谢病媒传播疾病的动物资源科司，特别是安德烈彼得森，丽莎马苏迪，贝尔纳奥布莱恩，约翰·利德尔的老鼠和兔子的关心和维护。我们也想感谢艾米·乌尔曼，特里萨·拉塞尔，和Barbara J.约翰逊朝着这个手稿的贡献。最后，我们想承认艾丽萨埃克特通信在疾病预防控制中心副主任办公室指导拍摄这个手稿与所有相关的合法性产生的插图和朱迪·拉维尔。

1。幻灯片准备的淋巴集合 （电影1） <ol><li>动物和外用3％过氧化氢的地方，轻轻地从积极喂养蜱5分钟，然后在70％乙醇10分钟，到表面消毒。 </li><li>与PAP笔硅烷涂层显微镜幻灯片上绘制一个圆，并放置在PAP笔圈的刻度。 <ol><li>硅烷涂层幻灯片用最好的淋巴坚持显微镜幻灯片。 </li></ol></li><li>在解剖显微镜下（1X目标，10X目镜，放大倍率3.5X）蜱。 </li><li>用钳子轻轻地往下推，瘫倒蜱的腿和固定滴答滴答的背部。 </li></ol> 注：固定蜱时不要按太硬，因为这可能会破坏肠或穿刺蜱和污染淋巴。 <ol start="5"><li>截肢日蜱的腿或腿é远端关节用细点，一次性手术刀。要确定传染性，通过淋巴只有1腿需要截肢。为幻灯片上的淋巴集合几个腿被截肢。 </li></ol> 注：切不要太靠近身体的腿，因为这可能会导致肠污染的淋巴。 <ol start="6"><li>腿或腿被切断后，继续为淋巴分泌到幻灯片的腿轻轻涂抹蜱的背部的压力。轻轻移动在幻灯片上的刻度，周围传播的淋巴。 </li></ol> 2。唾液腺切除 （电影2及3） <ol><li>现场数25μL池磷酸盐缓冲液（PBS），并放置到显微镜玻片PBS的池成蜱。 </li><li>在解剖显微镜下（1X目标，10X目镜，放大倍率3.5X）蜱。 </li><li>与精尖夹钳稳定的刻度S由控股的基础上花序（口器）或蜱背面。 </li><li>插入蜱背面的细尖镊子和切片，蜱的背部暴露的器官。如果需要的肠可以被删除在这个时候，转移到新鲜的PBS在显微镜幻灯片池或含PBS到离心管。 </li><li>找到一双位于双边沿着腿蜱唾液腺（葡萄状簇）。如果唾液腺是不可见的，其中包括蜱碎片移动的主要刻度部分，仍然含有唾液腺，新鲜的PBS池，以减少唾液腺的破坏和损失。 </li></ol> 注：在喂食早些时候，唾液腺也很难找到，因为它们没有发达相比，后来在喂养。 <ol start="6"><li>删除与精尖的镊子和新鲜的PBS池发生的蜱唾液腺。 </li><li>无线网络日罚款放倒钳唾液腺转移到另一个干净的25μLPBS池，重复此清洗步骤3-4次，以消除任何外部的微生物和蜱碎片。 </li></ol> 注意：洗净，轻轻地减少损失和破坏个人的唾液腺唾液腺集群。 <ol start="8"><li>放入清洁池的PBS硅烷涂层幻灯片或含PBS在离心管的腺体。 </li></ol> 3。唾液收集 （动画4） <ol><li>轻轻地取出成年雌蜱从兔或其他主机采用精尖的镊子，就在他们下车完全充血，附件后约5-7天。 </li><li>坚持到显微镜幻灯片用透明胶带的一端近肿胀蜱。磁带应放在约四分之三向上剔的背部向头部的方式，留下蜱的基础花序（口器）暴露。</li><li>磁带凡符合蜱背表面吸管5％的毛果芸香碱溶液5μL（甲醇）的前缘。让磁带罗卡没有让罗卡接触蜱的基础花序，灯芯在蜱的背部。 </li><li>挂载到显微镜玻片无毒黏土一块约一英寸从蜱的口器。 </li><li>用细尖镊子折断，一把拉住毛细管28到所需直径的小费。 </li><li>在解剖显微镜下查看蜱的基础花序。 </li><li>轻轻放入上颌触须向驻留在毛细管外拉毛细管管蜱的hypostome的。 </li><li>按入泥毛细管的另一端举行的毛细管到位。 </li><li>将高湿度（例如，一个有盖的发泡胶盒内衬湿PAP内暗室安装垂涎三尺蜱<html>呃毛巾）。倾斜所以hypostome点到容器底部的幻灯片，让重力帮助收集唾液中。 </li><li>在室温下放置容器。 </li><li>密切监察垂涎三尺的第一个小时的蜱，并收集唾液，因为它是与巴斯德吸管灯泡的驱逐毛细管产生。在第一个小时后，检查每隔一小时至少4个小时的积累。继续收集，因为它产生的唾液。 </li></ol> 注：一旦足够的唾液可以停止正在执行的研究收集唾液采集。 <ol start="12"><li>如果蜱是不垂涎三尺，如果需要更多的唾液，唾液可偶尔使用毛细管按摩hypostome诱导。 </li><li>新增0.1唾液和存储在-80°C，直到所需的蛋白酶抑制剂的鸡尾酒卷。 </li></ol> 4。代表结果e_content“&gt;电影演示如何把握部分喂养的一肩胛若虫和截肢的腿，收集到显微镜玻片淋巴。一旦腿或腿截肢分泌流畅清晰（图1A和1B）。如果肠破裂淋巴出现浑浊，因为它是截肢（S）（图1C和1D）。 电影中的第2和第3若虫后，已为48或72小时喂食唾液腺提取证明。蜱被刺破后，通常是一个有很多杂物（包括气管，马氏管，血液，结缔组织等），以防止丢失或破坏唾液腺移动蜱新鲜的PBS池。图2A和2B显示唾液腺位于若虫已被切开后，图2c显示唾液腺集群中删除的PBS池。 唾液收集从一成立肩胛成年女性，我<html>在电影4和图3 S所示。蜱成毛细管垂涎三尺观察在电影5。这唾液收集方法用毛果芸香碱刺激唾液分泌，可收益率超过20％的成年女性蜱唾液微升。 <img alt="图1。" src="/files/ftp_upload/3894/3894fig1.jpg" /> 图1。结构的一肩胛若虫的标记。 电影1。 肩突硬蜱血淋巴收集。 <a href="http://www.jove.com/files/ftp_upload/3894/3894movie1.avi">点击这里看电影</a> 。 <img alt="图2。" src="/files/ftp_upload/3894/3894fig2.jpg" /> 图2。无公害（A和B）和污染（拆建）若虫的腿血淋巴渗出。 电影2。从48小时喂一唾液腺提取肩胛若虫。pload/3894/3894movie2.avi“&gt;点击这里看电影。 电影3。从72小时喂一唾液腺提取肩胛若虫。 <a href="http://www.jove.com/files/ftp_upload/3894/3894movie3.avi">点击这里看电影</a> 。 <img alt="图3。" src="/files/ftp_upload/3894/3894fig3.jpg" /> 图3。 肩突硬蜱若虫唾液腺。 （A和B）唾液腺的例子，在72小时喂食若虫，前提取。 （c）删除唾液腺集群。 电影4。唾液收集从一个成人一成立肩胛女性蜱。 <a href="http://www.jove.com/files/ftp_upload/3894/3894movie4.avi">点击这里看电影</a> 。 <img alt="图4。" src="/files/ftp_upload/3894/3894fig4.jpg" /> 从一成人的唾液收集图4。 肩胛女式Ticks。 （A和B）勾选安装其在拉底与unpulled由黏土举行的毛细管毛细管hypostome的幻灯片上。 （拆建）湿盒含有垂涎三尺成人一肩胛雌蜱。 电影5。 肩突硬蜱的女性蜱成毛细管垂涎三尺。 <a href="http://www.jove.com/files/ftp_upload/3894/3894movie5.avi">点击这里观看电影</a> 。

<ol>
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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=Borreliacidal+activity+of+saliva+of+the+tick+Amblyomma+americanum.">Borreliacidal activity of saliva of the tick Amblyomma americanum.</a> Med. Vet. Entomol. 19, 90-95 (2005).</li><li>Ribeiro, J. M., Zeidner, N. S., Ledin, K., Dolan, M. C., Mather, T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+much+pilocarpine+contaminates+pilocarpine-induced+tick+saliva?.">How much pilocarpine contaminates pilocarpine-induced tick saliva?.</a> Med. Vet. Entomol. 18, 20-24 (2004).</li><li>Barker, R. W., Burris, E., Sauer, J. R., Hair, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composition+of+tick+oral+secretions+obtained+by+three+different+collection+methods.">Composition of tick oral secretions obtained by three different collection methods.</a> J. Med. Entomol. 10, 198-201 (1973).</li><li>Burgdorfer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemolymph+test.+A+technique+for+detection+of+rickettsiae+in+ticks.">Hemolymph test. A technique for detection of rickettsiae in ticks.</a> Am. J. Trop. Med. Hyg. 19, 1010-1014 (1970).</li></ol>

我们什么都没有透露。

蜱血淋巴，唾液腺和唾液的收集是非常重要的研究蜱传病原体传播，流行，传播，扩散，并在双方的滴答和主机6,11-13,20,23,29的持久性。有几种方法剖析蜱30,31的 。然而，当收集唾液腺关键是正确剖析蜱唾液腺不破裂，或在蜱的遗体丢失。一旦被删除要洗几次，清除肠污染，然后可以固定获得唾液腺提取物（SGE）染色或PBS地面幻灯片上，他们需要从蜱唾液腺。上海黄金交易所是更?...

<table border="1"><tbody><tr><td> 试剂 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>过氧化氢</td><td>费舍尔</td><td> H312-500 </td></tr><tr><td>乙醇</td><td>阿库罗斯福冈</td><td> 61509-5000 </td></tr><tr><td> PBS </td><td>波士顿生物制品</td><td> BM-2205 </td></tr><tr><td>杜蒙精细镊子（3C） </td><td>费舍尔</td><td> NC9906085 </td></tr><tr><td>硅烷偶联剂处理的载玻片</td><td> bioworld </td><td> 42763007-1 </td></tr><tr><td>子宫颈抹片笔</td><td> bioworld </td><td> 21750008-1 </td></tr><tr><td>超级霜加显微镜载玻片</td><td>费舍尔</td><td> 12-550-18 </td></tr><tr><td>匹罗卡品</td><td>西格玛</td><td> P6503-5G </td></tr><tr><td>蛋白酶抑制剂</td><td>西格玛</td><td> P2714 </td></tr><tr><td> ＃11一次性手术刀</td><td>羽毛</td><td> 2975＃ </td></tr><tr><td>无毒橡皮泥</td><td&gt;费舍尔</td><td> S17307 </td></tr><tr><td>毛细管</td><td>大通科研玻璃，公司</td><td> 40A502 </td></tr></tbody></table>

saliva, salivary gland, and hemolymph collection from ixodes scapularis ticks

蜱遍布世界各地，并与许多蜱传疾病的折磨人类。蜱是莱姆病和蜱传回归热（ 伯氏菌），洛矶山斑疹热（ 立克次氏体 ），埃立克体病（ 查菲埃立克体和E.球菌 ），无形体病（ 嗜吞噬细胞无形体 ），脑炎（蜱的病原体引起的载体森林脑炎病毒），巴贝斯虫病（ 巴贝斯虫属），科罗拉多蜱热（Colti病毒），兔热病（ 弗朗西斯tularensis）1-8。要正确传输到主机，这些传染性病原体的差异调节基因表达，与蜱蛋白质交互，并通过蜱3,9-13迁移。例如，莱姆病代理， 莱姆病 ，蜱的地方性周期14,15盛宴和饥荒阶段适应，通过基因差异表达。此外，作为硬蜱蜱消耗吸血伯从肠到的hemocoel，他们前往唾液腺，并传送到主机与被驱逐的唾液9,16-19复制和迁移。 由于蜱送入主机通常响应与一个强大的止血和先天免疫反应11,13,20-22。尽管这些宿主反应， 一肩胛可以养活了好几天，因为蜱唾液中含有的免疫调节的蛋白质，溶解剂，抗凝血剂，并fibrinolysins帮助蜱喂养3,11,20,21,23。由蜱唾液或唾液腺提取物（SGE）具有免疫调节活动方便，众多的蜱传病原体3,20,24-27的传输，扩散和传播。为了进一步了解如何蜱传播的传染性病原体引起的疾病，它是必不可少的积极剖析饲养蜱和收集蜱唾液。这个视频协议演示的夹层技术从积极喂养一血淋巴的收集和唾液腺的去除肩胛若虫后，48和72小时后鼠标的位置。我们还表明，从一成年女性唾液收集肩胛蜱。

Watch this Scientific Journal Video about Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks at JoVE.com

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

蜱血淋巴感染，唾液腺和唾液的收集是非常重要的研究蜱传病原体引起疾病。在这个协议中，我们演示了如何收集喂血淋巴和唾液腺<em&gt;肩突硬蜱</em&gt;若虫。我们还表明，从女性的唾液收集<em&gt;一。肩胛</em&gt;成年人。

唾液，唾液腺，淋巴收集肩突硬蜱蜱

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

saliva-salivary-gland-hemolymph-collection-from-ixodes-scapularis

Research

JoVE Journal

Immunology and Infection

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

29.2K 次观看.  Centers for Disease Control and Prevention. 蜱血淋巴感染，唾液腺和唾液的收集是非常重要的研究蜱传病原体引起疾病。在这个协议中，我们演示了如何收集喂血淋巴和唾液腺肩突硬蜱若虫。我们还表明，从女性的唾液收集一。肩胛成年人。

视频: Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

RNA Interference in Ticks

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors of human diseases1 and the most important vectors affecting cattle industry worldwide2. Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodida and are distributed worldwide from Arctic to tropical regions3. Despite efforts to control tick infestations, these ectoparasites remain a serious problem for human and animal health4,5.
RNA interference (RNAi)6 is a nucleic acid-based reverse genetic approach that involves disruption of gene expression in order to determine gene function or its effect on a metabolic pathway. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger7-9. Post-transcriptional gene silencing mechanisms initiated by dsRNA have been discovered in all eukaryotes studied thus far, and RNAi has been rapidly developed in a variety of organisms as a tool for functional genomics studies and other applications10.
RNAi has become the most widely used gene-silencing technique in ticks and other organisms where alternative approaches for genetic manipulation are not available or are unreliable5,11. The genetic characterization of ticks has been limited until the recent application of RNAi12,13. In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens14. Herein, a method for RNAi through injection of dsRNA into unfed ticks is described. It is likely that the knowledge gained from this experimental approach will contribute markedly to the understanding of basic biological systems and the development of vaccines to control tick infestations and prevent transmission of tick-borne pathogens15-19.

A method for RNA interference (RNAi) by injection of dsRNA into unfed ticks is described. RNAi is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulation has been limited.

RNA干扰在蜱

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors ...

科研

免疫与感染

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes. Investigation of transcripts and gene regulators that are expressed in tissues in which the mosquito host and pathogen interact, and in organs involved in reproduction are of great interest for strategies to reduce mosquito-borne disease transmission and disrupt egg development. A number of tools have been employed to study and validate the temporal and tissue-specific regulation of gene expression. Here, we describe protocols that have been developed to obtain spatial information, which enhances our understanding of where specific genes are expressed and their products accumulate. The protocol described has been used to validate expression and determine accumulation patterns of transcripts in tissues related to mosquito-borne pathogen transmission, such as female salivary glands, as well as subcellular compartments of ovaries and embryos, which relate to mosquito reproduction and development. 
 The following procedures represent an optimized methodology that improves the efficiency of various steps in the protocol without loss of target-specific hybridization signals. Guidelines for RNA probe preparation, dissection of soft tissues and the general procedure for fixation and hybridization are described in Part A, while steps specific for the collection, fixation, pre-hybridization and hybridization of mosquito embryos are detailed in Part B.

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.

杂交<em&gt;原位</em&gt;和蚊虫唾液腺，卵巢，胚胎

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.

Lyme disease is the most commonly-reported vector-borne disease in North America. The causative agent, Borrelia burgdorferi is a spirochete bacterium transmitted by Ixodid ticks. Transmission and detection of infection in animal models is optimized by the use of tick feeding, which we describe here.

蜱对动物饲养的传输和Xenodiagnosis在莱姆病研究

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the  attachment and blood feeding of Ixodes species ticks on ...

Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice

Ixodes scapularis, the vector of Lyme disease, is one of the most important disease vectors in the eastern and Midwestern United States. This species is a three host tick that requires a blood meal from a vertebrate host for each development stage, and the adult females require a blood meal for reproduction. Larval ticks attach to their host for 3 - 5 days for feeding and drop off the host when fully engorged. This dependency on several different hosts and the lengthy attachment time for engorgement complicates tick rearing in the laboratory setting. However, to understand tick biology and tick-pathogen interactions, the production of healthy, laboratory-reared ticks is essential. Here, we demonstrate a simple, cost-effective protocol for immature tick feeding on mice. We modified the existing protocols for decreased stress on mice and increased tick feeding success and survival by using disposable cages without mesh bottoms to avoid contact of ticks with water contaminated with mice urine and feces.

Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice

The production of healthy laboratory-reared ticks is essential to studies on tick biology, and tick-pathogen interactions. Here we demonstrate a simple protocol for immature tick feeding that is cost-effective and less stressful to mice.

饲养<em&gt;肩突硬蜱，</em&gt;黑腿蜱：饲养对小鼠未成熟阶段

Ixodes scapularis, the vector of Lyme disease, is one of the most important disease vectors in the eastern and Midwestern United States. This species is a ...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

小鼠涎腺功能的测定方法

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, including cell biology, oncology, dentistry, and immunology. The SMG is an exocrine gland comprised of secretory epithelial cells, myofibroblasts, endothelial cells, nerves, and extracellular matrix. Dynamic cellular processes in the rat and mouse SMG have previously been imaged, mostly using inverted multi-photon microscope systems. Here, we describe a straightforward protocol for the surgical preparation and stabilization of the murine SMG in anesthetized mice for in vivo imaging with upright multi-photon microscope systems. We present representative intravital image sets of endogenous and adoptively transferred fluorescent cells, including the labeling of blood vessels or salivary ducts and second harmonic generation to visualize fibrillar collagen. In sum, our protocol allows for surgical preparation of mouse salivary glands in upright microscopy systems, which are commonly used for intravital imaging in the field of immunology.

We describe a protocol to surgically expose and stabilize the murine submandibular salivary gland for intravital imaging using upright intravital microscopy. This protocol is easily adaptable to other exocrine glands of the head and neck region of mice and other small rodents.

直立活体显微术制备小鼠颌下腺涎腺

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, ...

Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to restore function of glands damaged by radiation therapies or due to pathologies associated with Sj&#246;gren's syndrome. A second goal of researchers is to define the mechanisms by which viruses replicate within glandular tissue where they can then gain access to salivary fluids important for horizontal transmission. These goals highlight the need for a robust and accessible in vitro salivary gland model that can be utilized by researchers interested in the above mentioned as well as related research areas. Here we discuss a simple protocol to isolate epithelial cells from human salivary glands and propagate them in vitro. Our protocol can be further optimized to meet the needs of individual studies. Briefly, salivary tissue is mechanically and enzymatically separated to isolate single cells or small clusters of cells. Selection for epithelial cells occurs by plating onto a basement membrane matrix in the presence of media optimized to promote epithelial cell growth. These resulting cultures can be maintained as three-dimensional clusters, termed "salispheres", or grown as a monolayer on treated plastic tissue culture dishes. This protocol results in the outgrowth of a heterogenous population of mainly epithelial cells that can be propagated for 5-8 passages (15-20 population doublings) before undergoing cellular senescence.

We present a method for isolating and cultivating primary human salivary gland-derived epithelial cells. These cells exhibit gene expression patterns consistent with them being of salivary epithelial origin and can be grown as salispheres on basement membrane matrices derived from Engelbreth-Holm-Swarm tumor cells or as monolayers on treated culture dishes.

从人类唾液腺中分离唾液表皮细胞,作为脂质层或单层体进行体外生长

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

高产细胞外囊泡制剂的纯化远离病毒

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight loss, depreciation of the animals&#39; leather and also can act as a vector of several pathogens. Due to the exorbitant costs to control these parasites, damage to the environment caused by the inappropriate use of chemical acaricides, and the increased resistance against traditional parasiticides, alternative control of ticks, by the use of entomopathogenic fungi, for example, has been considered an interesting approach. Nevertheless, few studies have demonstrated how the tick&#39;s immune system acts to fight these entomopathogens. Therefore, this protocol demonstrates two methods used for entomopathogen inoculation into engorged females and two techniques used for hemolymph collection and hemocytes harvesting. Inoculation of pathogens at the leg insertion in the tick female&#39;s body allows evaluation of females biologic parameters unlike the inoculation between the scutum and capitulum, which frequently damages Gen&#233;&#39;s organ. Dorsal hemolymph collection yielded a higher volume recovery than collection through the legs. Some limitations of tick hemolymph collection and processing include i) high rates of hemocytes&#39; disruption, ii) hemolymph contamination with disrupted midgut, and iii) low hemolymph volume recovery. When hemolymph is collected through the leg cutting, the hemolymph takes time to accumulate at the leg opening, favoring the clotting process. In addition, fewer hemocytes are obtained in the collection through the leg compared to the dorsal collection, even though the first method is considered easier to be performed. Understanding the immune response in ticks mediated by entomopathogenic agents helps to unveil their pathogenesis and develop new targets for tick control. The inoculation processes described here require very low technological resources and can be used not only to expose ticks to pathogenic microorganisms. Similarly, the collection of tick hemolymph may represent the first step for many physiological studies.

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Analysis of tick hemolymph represents an important source of information on how some pathogens cause disease and how ticks immunologically respond to this infection. The present study demonstrates how to inoculate fungal propagules and collect hemolymph from Rhipicephalus microplus engorged females.

在无脊椎动物病理学研究中发现血淋巴收集和接种转移到大黄微加分中

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight ...

小鼠下颌下腺再生的实时实时成像

Interrogating Cell-Cell Interactions in the Salivary Gland via Ex Vivo Live Cell Imaging

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal role played by macrophages in driving the regenerative response. However, our understanding of this critical role has primarily relied on static views obtained from fixed tissue biopsies. To overcome this limitation and gain insights into these interactions in real time, this study outlines a comprehensive protocol for culturing salivary gland tissue ex vivo and capturing live images of cell migration.
The protocol involves several key steps: First, mouse submandibular salivary gland tissue is carefully sliced using a vibratome and then cultured at an air-liquid interface. These slices can be intentionally injured, for instance, through exposure to radiation, to induce cellular damage and trigger the regenerative response. To track specific cells of interest, they can be endogenously labeled, such as by utilizing salivary gland tissue collected from genetically modified mice where a particular protein is marked with green fluorescent protein (GFP). Alternatively, fluorescently-conjugated antibodies can be employed to stain cells expressing specific cell surface markers of interest. Once prepared, the salivary gland slices are subjected to live imaging using a high-content confocal imaging system over a duration of 12 h, with images captured at 15 min intervals. The resulting images are then compiled to create a movie, which can subsequently be analyzed to extract valuable cell behavior parameters. This innovative method provides researchers with a powerful tool to investigate and better understand macrophage interactions within the salivary gland following injury, thereby advancing our knowledge of the regenerative processes at play in this dynamic biological context.

作者聚焦：研究损伤后唾液腺再生中巨噬细胞-上皮细胞的相互作用 

Immunofluorescent imaging is constrained by the ability to observe complex, time-dependent biological processes in just a single snapshot in time. This study outlines a live-imaging approach conducted on precision-cut mouse submandibular gland slices. This approach allows for the real-time observation of cell-cell interactions during homeostasis and the processes of regeneration and repair.

通过离体活细胞成像询问唾液腺中的细胞间相互作用

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal ...