Os autores gostariam de agradecer à Divisão de Vector Borne-Doenças Animais Branch Resources, especificamente Andrea Peterson, Lisa Massoudi, Verna O&#39;Brien, e John Liddell para seu cuidado e manutenção dos ratos e coelhos. Gostaríamos também de agradecer a Amy Ullmann, Theresa Russell, e Barbara J. Johnson para suas contribuições para este manuscrito. Finalmente, gostaríamos de reconhecer Alissa Eckert no Gabinete do Diretor Adjunto de Comunicação do CDC para produzir as ilustrações gráficas e Judy Lavelle para dirigir todos os assuntos legais relacionados com a filmagem deste manuscrito.

1. Coleta de hemolinfa para a preparação de slides (Filme 1) <ol><li> Remover suavemente activamente carrapatos de alimentação a partir de um animal e em lugar de peróxido de hidrogénio a 3% tópica durante 5 minutos e, em seguida, em etanol a 70% durante 10 minutos à superfície esterilizar. </li><li> Com uma caneta pap desenhar um círculo sobre uma lâmina de microscópio com revestimento de silano e coloque o carrapato dentro do círculo caneta pap. <ol><li> Silano lâminas revestidas são utilizados para melhor adesão da hemolinfa para a lâmina de microscópio. </li></ol></li><li> Ver o carrapato sob um microscópio de dissecação (1X objetivo, ocular de 10X, a ampliação 3.5X). </li><li> Com cuidado, empurre para baixo no dorso do carrapato com uma pinça para afunilar as pernas do carrapato e imobilizar o carrapato. </li></ol> Nota: Quando imobilizar o carrapato não pressionar muito difícil, porque isso pode prejudicar o intestino ou perfure o carrapato e contaminar a hemolinfa. <ol start="5"><li> Amputar a perna do carrapato ou pernas em diaconjunto e distal com uma ponta fina descartável bisturi. Para determinar a infectividade via hemolinfa apenas 1 perna precisa ser amputado. Para a coleta de hemolinfa sobre uma lâmina várias pernas pode ser amputado. </li></ol> Nota: Não corte a perna muito perto do corpo, porque isso pode causar a contaminação do intestino médio da hemolinfa. <ol start="6"><li> Após a perna ou pernas são cortadas continuar a aplique uma pressão suave dorso do carrapato para a hemolinfa para secretar fora das pernas para o slide. Mova suavemente o carrapato em torno sobre o slide para espalhar a hemolinfa. </li></ol> 2. Remoção completa da glândula salivar (Filmes 2 e 3) <ol><li> Spot de vários pools 25 ul de tampão fosfato salino (PBS) numa lâmina de microscópio e colocar uma carraça em uma das piscinas PBS. </li><li> Ver o carrapato sob um microscópio de dissecação (1X objetivo, ocular de 10X, a ampliação 3.5X). </li><li> Estabilizar o carrapato com pinça ponta finas, segurando o capitulum base (partes da boca) ou a parte traseira da carraça. </li><li> Inserir a pinça de ponta fina na parte traseira do carrapato e fatiar o carrapato é dorso que expõem os órgãos. Se desejado, o intestino médio pode ser removido neste momento e transferido para um pool de fresco de PBS numa lâmina de microscópio ou para um tubo de microcentrífuga contendo PBS. </li><li> Encontre o par de glândulas salivares (uva-como clusters) localizados bilateralmente ao lado das pernas do carrapato. Se as glândulas salivares não são visíveis entre os detritos carrapato mover a porção de carraça principal, ainda contendo as glândulas salivares, a um pool de fresco de PBS para reduzir a ruptura ea perda das glândulas salivares. </li></ol> Nota: No início de uma alimentação, as glândulas salivares são mais difíceis de localizar, porque não são tão desenvolvido como comparado com mais tarde na alimentação. <ol start="6"><li> Retire as glândulas salivares do carrapato com uma pinça com pontas finas e coloque em uma piscina nova de PBS. </li><li> Wiª pinça de ponta fina transferir as glândulas salivares para outro limpo piscina PBS 25 ul, repita esta etapa de lavagem 3-4 vezes mais para remover todos os microrganismos externos e os restos do carrapato. </li></ol> Nota: Lave os aglomerados de glândulas salivares com cuidado para reduzir a perda e ruptura das glândulas salivares individuais. <ol start="8"><li> Colocar as glândulas em uma piscina limpa de PBS numa lâmina de silano revestido ou em um tubo de microcentrífuga contendo PBS. </li></ol> 3. Coleta de saliva (Movie 4) <ol><li> Retire com cuidado fêmeas adultas do coelho ou outro host usando uma pinça com pontas finas, pouco antes que eles saem totalmente ingurgitadas, cerca de 5-7 dias pós-fixação. </li><li> Respeite o carrapato quase ingurgitadas em uma extremidade de uma lâmina de microscópio com fita adesiva. A fita deve ser colocado cerca de ¾ do caminho para cima do dorso do carrapato em direção à cabeça, deixando capitulum o carrapato base (partes da boca) exposta.</li><li> Sempre que a fita se encontra com o bordo anterior do dorsal superfície carrapatos uL de solução de pipeta 5 pilocarpina 5% (em metanol). Permitir a fita para o pavio pilocarpina sobre o carrapato do dorso, sem permitir que a pilocarpina para entrar em contato com o carrapato capitulum base. </li><li> Montar uma peça de non-toxic massa de modelar para a lâmina de microscópio aproximadamente uma polegada de peças bucais do carrapato. </li><li> Usando finas fórceps com pontas romper a ponta de um tubo capilar 28 puxado para o diâmetro desejado. </li><li> Ver capitulum o carrapato com base em um microscópio de dissecação. </li><li> Suavemente encaixar hypostome do carrapato para dentro do tubo capilar puxado permitindo que os palpos maxilares a residir no exterior do tubo capilar. </li><li> Pressione a extremidade oposta do tubo capilar na argila para segurar o tubo capilar no lugar. </li><li> Coloque o carrapato montado salivando dentro de uma câmara escura, com umidade elevada (por exemplo, uma caixa de isopor com tampa forrada com pap molhado<html> toalhas de ER). Inclinar a lâmina de modo que os pontos hypostome para o fundo do recipiente, permitindo que a gravidade para ajudar na recolha de saliva. </li><li> Colocar o recipiente à temperatura ambiente. </li><li> Acompanhar de perto os carrapatos salivando para a primeira hora, e recolher saliva, como é gerado por expulsando-o para fora dos tubos capilares com uma lâmpada de pipeta de Pasteur. Após a primeira hora, vá acumulação cada hora durante pelo menos 4 horas. Continuar a coleta de saliva como ele é gerado. </li></ol> Nota: aquisição de saliva pode ser interrompida uma vez bastante saliva são coletadas para o estudo que está sendo executada. <ol start="12"><li> Se os carrapatos não estão salivando ou se mais saliva é necessária, salivação pode ocasionalmente ser induzida com o uso do tubo capilar para massagear a hypostome. </li><li> Adicionar 0,1 volumes de cocktail inibidor de protease para a saliva e armazenar a -80 ° C até serem necessários. </li></ol> 4. Os resultados representativos e_content &quot;&gt; Movie 1 demonstra como a realização de uma ninfa parcialmente alimentado I. scapularis e amputar as pernas para coletar hemolinfa sobre uma lâmina de microscópio. Uma vez que a perna ou pernas amputadas um líquido claro é secretada (Figura 1A e 1B). Se o intestino é rompida na hemolinfa parece turvo como é sai da perna amputada (s) (Figura 1C e 1D). A extracção de glândulas salivares após a ninfa tem alimentado durante 48 ou 72 horas é demonstrado nos filmes 2 e 3. Após a marcação é perfurada há geralmente uma grande quantidade de detritos (consistindo de traqueia, malpighiano túbulos, sangue, tecido conjuntivo, etc), para impedir a perda ou a ruptura das glândulas salivares mover o carrapato a um pool de fresco de PBS. As Figuras 2A e 2B mostram onde as glândulas salivares estão localizadas após a ninfa foi cortado e aberto Figura 2C mostra removido aglomerados de glândulas salivares numa piscina de PBS. Coleta de saliva criada a partir de I. scapularis fêmeas adultas i<html> s mostrado na filme 4 e figura 3. Um carrapato salivando em um tubo capilar é observado em filme 5. Este método de coleta da saliva usada para estimular a salivação de pilocarpina e pode render mais de 20 ul de saliva por carrapato fêmea adulta. <img alt="Figura 1." src="/files/ftp_upload/3894/3894fig1.jpg" /> Figura 1. Estruturas rotuladas de uma ninfa scapularis I.. Filme 1. Ixodes scapularis coleção hemolinfa. <a href="http://www.jove.com/files/ftp_upload/3894/3894movie1.avi">Clique aqui para assistir filme</a> . <img alt="Figura 2." src="/files/ftp_upload/3894/3894fig2.jpg" /> Figura 2. Não contaminadas (A &amp; B) e contaminado (C &amp; D) hemolinfa exalando da perna da ninfa. Filme 2. Extração da glândula salivar de um 48 horas alimentados I. ninfa scapularis.pload/3894/3894movie2.avi &quot;&gt; Clique aqui para assistir filme. Filme 3. Extração da glândula salivar de um 72 horas alimentados I. ninfa scapularis. <a href="http://www.jove.com/files/ftp_upload/3894/3894movie3.avi">Clique aqui para assistir filme</a> . <img alt="Figura 3." src="/files/ftp_upload/3894/3894fig3.jpg" /> Figura 3. Glândulas salivares Ixodes scapularis ninfa. (A &amp; B) Exemplos de glândulas salivares em um 72 horas alimentados ninfa, antes da extração. (C) removido do cluster da glândula salivar. Movie 4. Coleta de saliva criado a partir de um adulto I. carrapato fêmea scapularis. <a href="http://www.jove.com/files/ftp_upload/3894/3894movie4.avi">Clique aqui para assistir filme</a> . <img alt="Figura 4." src="/files/ftp_upload/3894/3894fig4.jpg" /> Figura 4. Coleta de saliva de adultos de I. scapularis t mulheresicks. (A e B) Assinale montado sobre uma lâmina com a sua hypostome no final puxado do tubo capilar, com o fim unpulled do tubo capilar realizada por argila de modelagem. (C &amp; D) câmara úmida contendo salivando adulto I. scapularis carrapatos fêmeas. Filme 5. Ixodes scapularis carrapato fêmea salivando em um tubo capilar. <a href="http://www.jove.com/files/ftp_upload/3894/3894movie5.avi">Clique aqui para assistir filme</a> .

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Não temos nada a divulgar.

A coleta de hemolinfa do carrapato, glândulas salivares e saliva é importante no estudo da transmissão de patógenos transmitida por carrapatos, a divulgação, prevalência, proliferação e persistência, tanto carrapato e do exército 6,11-13,20,23,29. Existem várias maneiras de dissecar um carrapato 30,31. No entanto, ao coletar as glândulas salivares é fundamental para dissecar o carrapato corretamente para as glândulas salivares não são rompidas ou perdido em restos do carrapato. Uma...

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

Carrapatos são encontrados em todo o mundo e afligem os seres humanos com muitas doenças transmitidas por carrapatos. Os carrapatos são vetores de patógenos que causam a doença de Lyme e transmitida por carrapatos febre recorrente (Borrelia spp.), Febre maculosa (Rickettsia rickettsii), erliquiose (Ehrlichia chaffeensis e E. equi), anaplasmose (Anaplasma phagocytophilum), encefalite (tick- vírus da encefalite transmitida), babesiose (Babesia spp.), Colorado febre do carrapato (Coltivirus), e tularemia (Francisella tularensis) 1-8. Para ser adequadamente transmitida para o anfitrião destes agentes infecciosos diferencialmente regulam a expressão gênica, interagir com as proteínas de carrapatos, e migram através do carrapato 3,9-13. Por exemplo, o agente da doença de Lyme, Borrelia burgdorferi, adapta-se através da expressão diferencial de genes para as fases de festa e fome de ciclo enzoótico do carrapato 14,15. Além disso, como carrapatos um Ixodes consome umafarinha de sangue Borrelia replicar e migrar do intestino para a hemocele, onde eles viajam para as glândulas salivares e são transmitidos para o host com a saliva expelida 9,16-19. Como um carrapato se alimenta do hospedeiro geralmente responde com uma resposta hemostática e inata imunológico forte 11,13,20-22. Apesar destas respostas de acolhimento, I. scapularis pode se alimentar por vários dias porque a saliva do carrapato contém proteínas que são imunomoduladores, agentes líticos, anticoagulantes, e fibrinolysins para ajudar o carrapato alimentando 3,11,20,21,23. As atividades imunomoduladoras possuídos pela saliva do carrapato ou extrato de glândula salivar (SGE) facilitar a transmissão, a proliferação e disseminação de inúmeros carrapatos patógenos 3,20,24-27. Para entender melhor como carrapatos agentes infecciosos causam a doença é essencial para dissecar alimentando ativamente carrapatos e coletar a saliva do carrapato. Este protocolo de vídeo demonstra técnicas de dissecação paraa recolha de hemolinfa e à remoção das glândulas salivares de alimentação activamente I. scapularis ninfas após 48 e 72 horas pós posicionamento do mouse. Nós também demonstramos a coleta da saliva de uma fêmea adulta I. carrapato scapularis.

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

A coleta de hemolinfa do carrapato infectado, glândulas salivares e saliva é importante estudar como a transmitida por carrapatos patógenos causam doenças. Neste protocolo, demonstramos como coletar glândulas salivares e hemolinfa de alimentação<em&gt; Ixodes scapularis</em&gt; Ninfas. Nós também demonstramos a coleta da saliva da fêmea<em&gt; I. scapularis</em&gt; Adultos.

A saliva, glândula salivar, e Coleta de hemolinfa de carrapatos Ixodes scapularis

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 visualizações.  Centers for Disease Control and Prevention. A coleta de hemolinfa do carrapato infectado, glândulas salivares e saliva é importante estudar como a transmitida por carrapatos patógenos causam doenças. Neste protocolo, demonstramos como coletar glândulas salivares e hemolinfa de alimentação Ixodes scapularis Ninfas. Nós também demonstramos a coleta da saliva da fêmea I. scapularis Adultos.

Vídeo: 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 de interferência em Carrapatos

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

Imunologia e Infecção

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.

Hibridização<em&gt; In situ</em&gt; Das glândulas salivares, ovários e embriões de mosquitos vetores

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.

Alimentação dos Carrapatos em Animais de Transmissão e Xenodiagnóstico em 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 ...

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.

Criação<em&gt; Ixodes scapularis,</em&gt; O Ticked Black-legged: Alimentando estágios imaturos em ratos

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.

Um método para a medição da função da glândula salivar em ratos

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.

Preparação de murino das glândulas salivares Submandibular para microscopia Intravital vertical

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.

Isolação de pilhas epithelial salivares das glândulas salivares humanas para o crescimento in vitro como Salispheres ou 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 ...

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.

Purificação de preparações de vesículas extracelulares de alto rendimento longe do vírus

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.

Divulgando a coleta de Hemolinfas e a inoculação de blastósporos de Metarhizium em carrapatos de Rhipicephalus MICROPLUS para estudos de patologia de invertebrados

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

Imagens ao vivo em tempo real da regeneração da glândula submandibular do camundongo

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.

Autor em destaque: Estudando as interações entre macrófagos e células epiteliais na regeneração da glândula salivar após lesão 

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.

Interrogando interações célula-célula na glândula salivar via imagem de células vivas ex vivo

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