The authors would like to thank the Division of Vector-Borne Diseases Animal Resources Branch, specifically Andrea Peterson, Lisa Massoudi, Verna O'Brien, and John Liddell for their care and maintenance of the mice and rabbits. We would also like to thank Amy Ullmann, Theresa Russell, and Barbara J. Johnson for their contributions toward this manuscript. Finally, we would like to acknowledge Alissa Eckert in the Office of the Associate Director for Communication at the CDC for producing the graphic illustrations and Judy Lavelle for directing all the legalities associated with the filming of this manuscript.

1. Hemolymph collection for slide preparation
(Movie 1)
<ol>
	<li>Gently remove actively feeding ticks from an animal and place in 3% topical hydrogen peroxide for 5 minutes and then in 70% ethanol for 10 minutes to surface sterilize.</li>
	<li>With a pap pen draw a circle onto a silane coated microscope slide and place the tick within the pap pen circle.
	<ol>
		<li>Silane coated slides are used for best adherence of the hemolymph to the microscope slide.</li>
	</ol>
	</li>
	<li>View the tick under a dissecting microscope (1X objective, 10X eyepiece, 3.5X magnification).</li>
	<li>Gently push down on the tick's dorsum with forceps to splay the tick's legs and immobilize the tick.</li>
</ol>
Note: When immobilizing the tick do not press too hard because this may disrupt the midgut or puncture the tick and contaminate the hemolymph.
<ol start="5">
	<li>Amputate the tick's leg or legs at the distal joint with a fine point disposable scalpel. To determine infectivity via hemolymph only 1 leg needs to be amputated. For collection of hemolymph on a slide several legs may be amputated.</li>
</ol>
Note: Do not cut the leg too close to the body because this may cause midgut contamination of the hemolymph.
<ol start="6">
	<li>After the leg or legs are cut continue to gently apply pressure to the tick's dorsum for the hemolymph to secrete out of the legs onto the slide. Gently move the tick around on the slide to spread the hemolymph.</li>
</ol>
2. Salivary gland removal
(Movies 2 &amp; 3)
<ol>
	<li>Spot several 25 &mu;l pools of phosphate buffered saline (PBS) onto a microscope slide and place a tick into one of the PBS pools.</li>
	<li>View the tick under a dissecting microscope (1X objective, 10X eyepiece, 3.5X magnification).</li>
	<li>Stabilize the tick with fine tipped forceps by holding the basis capitulum (mouth parts) or the rear of the tick.</li>
	<li>Insert the fine tipped forceps into the rear of the tick and slice up the tick's dorsum to expose the organs. If desired the midgut can be removed at this time and transferred to a fresh pool of PBS on a microscope slide or to a microfuge tube containing PBS.</li>
	<li>Find the pair of salivary glands (grape-like clusters) located bilaterally alongside the legs of the tick. If the salivary glands are not visible amongst the tick debris move the main tick portion, still containing the salivary glands, to a fresh pool of PBS to reduce the disruption and loss of the salivary glands.</li>
</ol>
Note: Earlier in a feeding, the salivary glands are harder to locate because they are not as developed as compared to later on in the feeding.
<ol start="6">
	<li>Remove the salivary glands from the tick with fine tipped forceps and place in a fresh pool of PBS.</li>
	<li>With fine tipped forceps transfer the salivary glands to another clean 25 &mu;l PBS pool, repeat this wash step 3-4 more times to remove any external microorganisms and tick debris.</li>
</ol>
Note: Wash the salivary gland clusters gently to reduce the loss and disruption of individual salivary glands.
<ol start="8">
	<li>Place the glands into a clean pool of PBS on a silane coated slide or in a microfuge tube containing PBS.</li>
</ol>
3. Saliva collection
(Movie 4)
<ol>
	<li>Gently remove adult female ticks from the rabbit or other host using fine tipped forceps just before they drop off fully engorged, approximately 5-7 days post-attachment.</li>
	<li>Adhere the nearly engorged tick onto one end of a microscope slide with scotch tape. The tape should be placed approximately &frac34; of the way up the tick's dorsum toward the head, leaving the tick's basis capitulum (mouth parts) exposed.</li>
	<li>Where the tape meets the anterior edge of the ticks dorsal surface pipet 5 &mu;l of 5% pilocarpine solution (in methanol). Allow the tape to wick the pilocarpine over the tick's dorsum, without allowing the pilocarpine to come in contact with the tick's basis capitulum.</li>
	<li>Mount a piece of non-toxic modeling clay onto the microscope slide approximately one inch from the tick's mouthparts.</li>
	<li>Using fine tipped forceps break off the tip of a pulled capillary tube28 to the desired diameter.</li>
	<li>View the tick's basis capitulum under a dissecting microscope.</li>
	<li>Gently fit the tick's hypostome into the pulled capillary tube allowing the maxillary palps to reside on the outside of the capillary tube.</li>
	<li>Press the opposite end of the capillary tube into the modeling clay to hold the capillary tube in place.</li>
	<li>Place the mounted salivating tick inside a dark chamber with high humidity (e.g., a lidded styrofoam box lined with wet paper towels). Tilt the slide so the hypostome points to the bottom of the container, allowing gravity to aid in saliva collection.</li>
	<li>Place the container at room temperature.</li>
	<li>Closely monitor the salivating ticks for the first hour, and collect saliva as it is generated by expelling it out of the capillary tubes with a Pasteur pipet bulb. After the first hour, check accumulation every hour for at least 4 hours. Continue to collect the saliva as it is generated.</li>
</ol>
Note: Saliva acquisition can be stopped once enough saliva is collected for the study being performed.
<ol start="12">
	<li>If the ticks are not salivating or if more saliva is required, salivation can occasionally be induced by using the capillary tube to massage the hypostome.</li>
	<li>Add 0.1 volumes of protease inhibitor cocktail to the saliva and store at -80&deg;C until needed.</li>
</ol>
4. Representative Results
Movie 1 demonstrates how to hold a partially fed I. scapularis nymph and amputate the legs to collect hemolymph onto a microscope slide. Once the leg or legs are amputated a clear fluid is secreted (figure 1A and 1B). If the midgut is ruptured the hemolymph appears cloudy as it is comes out of the amputated leg(s) (figure 1C and 1D).
The extraction of salivary glands after the nymph has been feeding for 48 or 72 hours is demonstrated in movies 2 and 3. After the tick is punctured there is generally a lot of debris (consisting of trachea, malpighian tubules, blood, connective tissue etc.), to prevent the loss or disruption of the salivary glands move the tick to a fresh pool of PBS. Figures 2A and 2B show where the salivary glands are located after the nymph has been cut open and figure 2C shows removed salivary gland clusters in a pool of PBS.
Saliva collection set up from I. scapularis adult females is shown in movie 4 and figure 3. A tick salivating into a capillary tube is observed in movie 5. This method of saliva collection used pilocarpine to stimulate salivation and can yield over 20 &mu;l of saliva per adult female tick.
<img alt="Figure 1." src="/files/ftp_upload/3894/3894fig1.jpg" /> 
Figure 1. Labeled structures of an I. scapularis nymph.
Movie 1. Ixodes scapularis hemolymph collection. <a href="https://www.jove.com/files/ftp_upload/3894/3894movie1.avi">Click here to watch movie</a>.
<img alt="Figure 2." src="/files/ftp_upload/3894/3894fig2.jpg" /> 
Figure 2. Uncontaminated (A &amp; B) and contaminated (C &amp; D) hemolymph exuding from the nymph's leg.
Movie 2. Salivary gland extraction from a 48 hour fed I. scapularis nymph.<a href="https://www.jove.com/files/ftp_upload/3894/3894movie2.avi">Click here to watch movie</a>.
Movie 3. Salivary gland extraction from a 72 hour fed I. scapularis nymph. <a href="https://www.jove.com/files/ftp_upload/3894/3894movie3.avi">Click here to watch movie</a>.
<img alt="Figure 3." src="/files/ftp_upload/3894/3894fig3.jpg" /> 
Figure 3. Ixodes scapularis nymph salivary glands. (A &amp; B) Examples of salivary glands in a 72 hour fed nymph, prior to extraction. (C) Removed salivary gland cluster.
Movie 4. Saliva collection set up from an adult I. scapularis female tick. <a href="https://www.jove.com/files/ftp_upload/3894/3894movie4.avi">Click here to watch movie</a>.
<img alt="Figure 4." src="/files/ftp_upload/3894/3894fig4.jpg" /> 
Figure 4. Saliva collection from adult I. scapularis female ticks. (A &amp; B) Tick mounted on a slide with its hypostome in the pulled end of the capillary tube with the unpulled end of the capillary tube held by modeling clay. (C &amp; D) Humidified chamber containing salivating adult I. scapularis female ticks.
Movie 5. Ixodes scapularis female tick salivating into a capillary tube. <a href="https://www.jove.com/files/ftp_upload/3894/3894movie5.avi">Click here to watch movie</a>.

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We have nothing to disclose.

The collection of tick hemolymph, salivary glands, and saliva is important in the study of tick-borne pathogen transmission, prevalence, dissemination, proliferation, and persistence in both the tick and the host 6,11-13,20,23,29. There are several ways to dissect a tick30,31. However, when collecting salivary glands it is critical to dissect the tick properly so the salivary glands are not ruptured or lost in the tick's remains. Once the salivary glands are removed from the tick they need to be was...

<table border="1">
 <tbody>
 <tr>
 <td>Reagent</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>Hydrogen peroxide</td>
 <td>Fisher</td>
 <td>H312-500</td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Acros </td>
 <td>61509-5000</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Boston Bioproducts</td>
 <td>BM-2205</td>
 </tr>
 <tr>
 <td>Dumont Fine forceps (3C)</td>
 <td>Fisher</td>
 <td>NC9906085</td>
 </tr>
 <tr>
 <td>Silane treated microscope slides</td>
 <td>Bioworld</td>
 <td>42763007-1</td>
 </tr>
 <tr>
 <td>Pap pen</td>
 <td>Bioworld</td>
 <td>21750008-1</td>
 </tr>
 <tr>
 <td>Super frost plus microscope slides</td>
 <td>Fisher</td>
 <td>12-550-18</td>
 </tr>
 <tr>
 <td>Pilocarpine</td>
 <td>Sigma</td>
 <td>P6503-5G</td>
 </tr>
 <tr>
 <td>Protease inhibitor cocktail</td>
 <td>Sigma</td>
 <td>P2714</td>
 </tr>
 <tr>
 <td>#11 disposable scalpel</td>
 <td>Feather</td>
 <td>2975#11</td>
 </tr>
 <tr>
 <td>Nontoxic modeling clay</td>
 <td>Fisher</td>
 <td>S17307</td>
 </tr>
 <tr>
 <td>Capillary tubes</td>
 <td>Chase scientific Glass, inc </td>
 <td>40A502</td>
 </tr>
</tbody>
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saliva, salivary gland, and hemolymph collection from ixodes scapularis ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

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

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

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

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

29.2K Views.  Centers for Disease Control and Prevention. The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Video: 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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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

Real-Time Live Imaging of Mouse Submandibular Gland Regeneration

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.

Author Spotlight: Studying Macrophage-Epithelial Cell Interactions in Salivary Gland Regeneration After Injury 

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.

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