The study was supported by a grant from National Institutes of Health, National Institute of Dental and Craniofacial Research (DE025030).

The protocol described below was approved by the Institutional Animal Care and Use Committee and it follows the ethical guidelines established by the National Institutes of Health. All data presented in this report were generated by using female mice that were 10-12 weeks of age. The following strains of mice were used: C57BL/6, BALB/c, DBA1, 129S, and (B6XA/J) F1. All mice were housed in barrier cages (5 animals per cage) in specific pathogen-free conditions and provided feed and water ad libitum.
1. Preparation
<ol>
	<li>To prepare a stock solution of pilocarpine hydrochloride, weigh 10 mg of the compound and dissolve it in 1.776 mL of sterile isotonic saline by vortexing, to give a stock solution of 5.63 mg/mL. There is no need to sterilize this solution. But if desired, filter it through a 0.2 &#181;M filter. Store this stock solution in multiple aliquots in a -80 oC freezer for up to 3 months. Each frozen stock is for single use. Once thawed, do not refreeze the pilocarpine solution.</li>
	<li>Take 0.6 mL microfuge tubes and carefully punch a small hole in the bottom of each tube with a heated 18-gauge needle. Set these tubes into 2 mL tubes. The tubes need not be sterile.</li>
	<li>Using a sharp, sterile razor blade, cut cylindrical absorbent swabs into pieces about 2 cm in length. Cut each 2 cm piece diagonally to give 2 conical shaped swabs. Place one swab in each of the 0.6 mL microfuge tubes.</li>
	<li>Weigh the 0.6 mL microfuge tube containing the dry swab.</li>
	<li>Transfer the mice to be studied into a new clean cage with water bottles. Keep the mice without food for at least 2 h prior to the start of saliva collection to prevent food particles from contaminating the saliva collected. 
	NOTE: It is not necessary to keep 1 mouse per cage for this procedure. However, the number of mice placed in each cage depends on the specific institution&#39;s rules and regulations for animal use.</li>
	<li>Just before the end of 2 h, prepare the working pilocarpine solution (0.0563 mg/mL) by diluting the stock solution 100x in sterile saline. It is not necessary to further filter sterilize this solution. Always keep the working pilocarpine solution on ice. 
	NOTE: Table 1 gives the amount of pilocarpine to be injected for a range of mouse body weights to give a final dose of 0.375 mg/kg body weight.</li>
</ol>
2. Procedure
<ol>
	<li>Weigh each mouse and determine the dose of the anesthetic mix for each mouse using Table 1. Inject the first mouse with the appropriate volume of the anesthetic mix by the intraperitoneal route. Set the timer for 2 min (most mouse strains tested in this protocol go to sleep by 2 min). Ensure that the mouse is properly anesthetized by lack of walking when placed on a flat surface. If the mouse is still walking, wait for additional 2 min before proceeding to the next step. Apply a drop of lubricant ophthalmic ointment to both eyes to prevent drying. 
	NOTE: In Table 1, the dose of the anesthetic mix is 0.007 mL/g body weight. The optimal range for this procedure is 0.006 to 0.008 mL/g body weight. However, after 4 min, if the mouse is still walking or exhibiting jerky movements, consult with the institutional veterinarian for appropriately increasing the dose of the anesthetic.</li>
	<li>Determine the dose of pilocarpine for the mouse using Table 1 and inject the appropriate amount by intraperitoneal route. Set the timer for 2 min and insert the mouse into the 50 mL restrainer tube until the head and ears stick out of the cut end. Place the tube at a 45 degree angle, with the head down, and the ventral surface facing upward. Tape the tube to the procedure board to keep it from moving. 
	NOTE: Table 1 provides dosage for mice more than 17 g, as this procedure has not been attempted on mice weighing below 17 g.</li>
	<li>At the end of 2 min, gently insert a closed pair of micro dissecting forceps into the mouth and lift the lower jaw upwards to open the mouth. Push the forceps 1-2 mm further into the mouth, making sure that the tongue is seen resting on the top arm of the forceps. With another pair of fine forceps, hold the pre-weighed dry swab close to its conical tip.</li>
	<li>Gently slide the conical tip of the swab into the mouth. Withdraw the forceps while leaving the tip of the swab in the oral cavity.</li>
	<li>Grasp the wider end of the swab extending outside of the mouth and rotate it to allow the maximum area of contact with the mouth. Keep the swab in this position for 15 min. 
	NOTE: This ensures that the swab remains in the mouth for the duration of the collection. Note that the conical tip of the swab acts as a wick and the wider portion of the swab remains outside the mouth.</li>
	<li>At the end of 15 min, gently rotate the swab to collect any saliva that has not been absorbed and place the wet swab in the 0.6 mL microfuge tube. Close the tube and set it in the 2 mL tube placed on ice. 
	NOTE: At this time, the oral mucosa will appear completely dry. The mouse can now be moved back into its cage for recovery from anesthesia.</li>
	<li>Transfer the mouse in the cage. Place moistened food pellets in the cage after saliva collection to help faster rehydration. 
	NOTE: The mice wake up within 10 min after the end of the collection. Subcutaneous injection of 0.2 mL pre-warmed isotonic saline may also be given to accelerate recovery.</li>
	<li>Proceed to the next mouse.&#160;All the mice should be monitored until they have completely recovered and are ambulatory.</li>
</ol>
3. Measurements
<ol>
	<li>At the end of all collections, weigh the 0.6 mL tubes with the wet swab. Calculate the difference between the wet weight and dry weight to get the weight of saliva produced. Place the 0.6 mL tube with saliva back into the 2-mL tube.</li>
	<li>Cut off the caps of the 2 ml tubes. Then, centrifuge the 2 mL tubes for 2 min at 7500 x g, at 4oC in a micro centrifuge to recover the saliva. Measure the volume of saliva obtained using a micropipette.</li>
	<li>Express the results as saliva weight (mg)10 over 15 min or as a ratio of saliva weight (mg)/mouse body weight (g). 
	NOTE: If the volume of saliva extracted out of the swab has been measured, express the results as the volume of saliva recovered (mL) or the ratio of saliva volume (mL)/mouse body weight (g).</li>
</ol>

<ol><li>Proctor, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+physiology+of+salivary+secretion%5BTitle%5D)+AND+%22Periodontol+2000%22%5BJournal%5D)">The physiology of salivary secretion.</a> Periodontol 2000. 70 (1), 11-25 (2016).</li>
<li>Fox, R. I. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/16039337">Sjögren's syndrome.</a> Lancet. 366 (9482), 321-331 (2005).</li>
<li>Eisbruch, A., Kim, H. M., Terrell, J. E., Marsh, L. H., Dawson, L. A., Ship, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Xerostomia+and+its+predictors+following+parotid-sparing+irradiation+of+head-and-neck+cancer%5BTitle%5D)+AND+%22Int+J+Radiat+Oncol+Biol+Phys%22%5BJournal%5D)">Xerostomia and its predictors following parotid-sparing irradiation of head-and-neck cancer.</a> Int J Radiat Oncol Biol Phys. 50 (3), 695-704 (2001).</li>
<li>Marmary, Y., Fox, P. C., Baum, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluid+secretion+rates+from+mouse+and+rat+parotid+glands+are+markedly+different+following+pilocarpine+stimulation%5BTitle%5D)+AND+%22Comp+Biochem+Physiol+A+Comp+Physiol%22%5BJournal%5D)">Fluid secretion rates from mouse and rat parotid glands are markedly different following pilocarpine stimulation.</a> Comp Biochem Physiol A Comp Physiol. 88 (2), 307-310 (1987).</li>
<li>Lin, A. L., Johnson, D. A., Wu, Y., Wong, G., Ebersole, J. L., Yeh, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measuring+short-term+gamma-irradiation+effects+on+mouse+salivary+gland+function+using+a+new+saliva+collection+device%5BTitle%5D)+AND+%22Arch+Oral+Biol%22%5BJournal%5D)">Measuring short-term gamma-irradiation effects on mouse salivary gland function using a new saliva collection device.</a> Arch Oral Biol. 46 (11), 1085-1089 (2001).</li>
<li>Scofield, R. H., Asfa, S., Obeso, D., Jonsson, R., Kurien, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunization+with+short+peptides+from+the+60-kDa+Ro+antigen+recapitulates+the+serological+and+pathological+findings+as+well+as+the+salivary+gland+dysfunction+of+Sjogren%27s+syndrome%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Immunization with short peptides from the 60-kDa Ro antigen recapitulates the serological and pathological findings as well as the salivary gland dysfunction of Sjogren's syndrome.</a> J Immunol. 175 (12), 8409-8414 (2005).</li>
<li>Deshmukh, U. S., Nandula, S. R., Thimmalapura, P. R., Scindia, Y. M., Bagavant, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activation+of+innate+immune+responses+through+Toll-like+receptor+3+causes+a+rapid+loss+of+salivary+gland+function%5BTitle%5D)+AND+%22J+Oral+Pathol+Med%22%5BJournal%5D)">Activation of innate immune responses through Toll-like receptor 3 causes a rapid loss of salivary gland function.</a> J Oral Pathol Med. 38 (1), 42-47 (2009).</li>
<li>Deshmukh, U. S., Ohyama, Y., Bagavant, H., Guo, X., Gaskin, F., Fu, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inflammatory+stimuli+accelerate+Sj%C3%B6gren%27s+syndrome-like+disease+in+%28NZB+x+NZW%29F1+mice%5BTitle%5D)+AND+%22Arthritis+Rheum%22%5BJournal%5D)">Inflammatory stimuli accelerate Sjögren's syndrome-like disease in (NZB x NZW)F1 mice.</a> Arthritis Rheum. 58 (5), 1318-1323 (2008).</li>
<li>Szczerba, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interaction+between+innate+immunity+and+Ro52-induced+antibody+causes+Sj%C3%B6gren%27s+syndrome-like+disorder+in+mice%5BTitle%5D)+AND+%22Ann+Rheum+Dis%22%5BJournal%5D)">Interaction between innate immunity and Ro52-induced antibody causes Sjögren's syndrome-like disorder in mice.</a> Ann Rheum Dis. 75 (3), 617-622 (2016).</li>
<li>Takakura, A., Moreira, T., Laitano, S., De Luca Júnior, L., Renzi, A., Menani, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Central+muscarinic+receptors+signal+pilocarpine-induced+salivation%5BTitle%5D)+AND+%22J+Dent+Res%22%5BJournal%5D)">Central muscarinic receptors signal pilocarpine-induced salivation.</a> J Dent Res. 82 (12), 993-997 (2003).</li>
<li>Yao, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lipopolysaccharide-induced+elevation+and+secretion+of+interleukin-1beta+in+the+submandibular+gland+of+male+mice%5BTitle%5D)+AND+%22Immunology%22%5BJournal%5D)">Lipopolysaccharide-induced elevation and secretion of interleukin-1beta in the submandibular gland of male mice.</a> Immunology. 116 (2), 213-222 (2005).</li>
<li>Knudsen, J., Nauntofte, B., Josipovic, M., Engelholm, S. A., Hyldegaard, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+isoflurane+anesthesia+and+pilocarpine+on+rat+parotid+saliva+flow%5BTitle%5D)+AND+%22Radiat+Res%22%5BJournal%5D)">Effects of isoflurane anesthesia and pilocarpine on rat parotid saliva flow.</a> Radiat Res. 176 (1), 84-88 (2011).</li>
<li>Kohrs, R., Durieux, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ketamine%3A+teaching+an+old+drug+new+tricks%5BTitle%5D)+AND+%22Anesth+Analg%22%5BJournal%5D)">Ketamine: teaching an old drug new tricks.</a> Anesth Analg. 87 (5), 1186-1193 (1998).</li>
<li>Cold Spring Harbor Protocols. Cold Spring Harb Protoc. , Available from:
 <a target="_blank" href="http://cshprotocols.cshlp.org/">http://cshprotocols.cshlp.org/</a> (2006).</li>
<li>Kozaki, T., Hashiguchi, N., Kaji, Y., Yasukouchi, A., Tochihara, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+saliva+collection+using+cotton+swab+on+cortisol+enzyme+immunoassay%5BTitle%5D)+AND+%22Eur+J+Appl+Physiol%22%5BJournal%5D)">Effects of saliva collection using cotton swab on cortisol enzyme immunoassay.</a> Eur J Appl Physiol. 107 (6), 743-746 (2009).</li>
</ol>

The authors have no conflicts of interest to disclose.

Saliva production is a complex process and is influenced by many factors. Therefore, measuring salivary gland function in laboratory animals can be a challenge. An additional challenge is that repeated measurements of salivary gland function in the same animal are required to establish the onset of disease or to demonstrate recovery following treatment.
The swab method described in this report is technically simple to perform with multiple options to represent the data. The results obtained are reproducible, with little inter-operator variation. The method can detect a reduction in saliva production in different models of salivary gland dysfunction. A significant advantage is the use of inert, highly absorbent polymers and efficient recovery of the saliva collected, allowing measurement of salivary biomolecules. Another advantage of this method is that it is possible to carry out this procedure simultaneously in up to 2 mice at a time by a single operator. This is significantly more efficient than some of the other methods, where it is performed in a single mouse at a time.
The variability in saliva production measurements have been the bane of all saliva measurement techniques. To minimize variability, it is important to adhere strictly to the protocol. Critical steps include: selecting the same time of the day for fasting the mice and collecting saliva, appropriate dosing of anesthetic and pilocarpine (table 1), and accurate weight records of dry and wet swabs. Careful time management is required to set up to 2 mice at a time for saliva collection.
As shown in Figure 3, the amount of saliva produced by mice is strain dependent. Thus, appropriate strain, age, and sex matched mice should be used as controls. Titration of the pilocarpine dosage, for the specific strain and the experimental condition being investigated, is highly recommended. Although a higher dose of pilocarpine beyond 0.5 mg/kg induces increased production of saliva, to evaluate glandular hypofunction, it is recommended to use a lower dose. This allows for the identification of even minor differences in saliva production between the diseased and control mice. Doses of 0.5 mg/kg body weight and 0.375 mg/kg body weight have been used successfully to demonstrate salivary gland hypofunction. However, it should be noted that certain experimental model systems may require shorter or longer periods of collection than that described in this protocol. This should be tested by each laboratory.
A limitation of this method, and most saliva measuring methods is the need for anesthesia. The report with the vacuum method does not include the use of anesthetic5. However, the absence of anesthesia induces varying levels of anxiety in the mice. They tend to struggle and bite the tubing, and that in turn, can influence saliva output and collection efficiency. Isoflurane anesthesia in rats induces a drop-in pilocarpine induced saliva production12. In contrast, ketamine increases bronchial and salivary secretions through sympathetic stimulation13. We and others have successfully used the ketamine and xylazine mixture as an anesthetic for measuring pilocarpine induced saliva production4,7,8,9. The recommended range for surgical anesthesia is 100-200 mg/kg of ketamine and 5-16 mg/kg of xylazine14. The dose of 0.006-0.008 mL/g body weight used in this method (corresponds to 60-80 mg/kg of ketamine and 6-8 mg/kg of xylazine) falls in the lower end of the recommended range and is sufficient for providing a consistent level of immobilization without deep anesthesia.
The swab method was established as an alternative to the routinely used micropipet method. The major disadvantage of the pipet method was the high inter-operator variability and the need to collect saliva from one mouse at a time. The swab method addresses both these problems. The benefits include limiting the number of animals needed to achieve power in statistical analyses and increased efficiency. Replacing cotton swabs with inert polymer swabs offer a better option since cotton swabs are known to impact quantitation of biologically active molecules15.
Given the simplicity of the swab method and its high reproducibility between different individuals, it is hoped that the swab method will facilitate better comparisons of mouse models between researchers in different laboratories and institutions.

The salivary glands produce saliva in response to a variety of neurological and mechanical stimuli1. The stimuli are carried through the sympathetic and parasympathetic nervous system to the adrenergic and cholinergic receptors in the gland. Pilocarpine is a cholinergic, para-sympathomimetic agent that acts predominantly on muscarinic receptors. In the salivary gland, it induces the production of saliva by acting on the muscarinic acetylcholine receptor M31. Saliva production following pilocarpine administration is an indicator of the ability of the salivary glands to respond to stimulation and is commonly used as a measure of salivary gland function.
Accurate measurement of saliva production is critical in the study of salivary gland diseases including Sj&#246;gren&#39;s syndrome2 and radiation injury following head and neck cancer treatment3. Several different methods have been developed to measure saliva production in rodents. These include direct cannulation of the excretory salivary duct4, the collection of saliva from the oral cavity under vacuum5, and collection using glass capillaries6 or a micropipette7,8,9. Direct cannulation of the salivary duct provides the most accurate and pure saliva. However, this is a technically challenging procedure, and the potential for causing ductal injury precludes repetitive saliva collections from the same animal. Collecting saliva under vacuum can lead to variable results due to drying of the saliva from the tube. This loss is further exaggerated in mice with reduced salivation. Glass capillaries allow the collection of saliva for subsequent analyses, however, changes in viscosity of the saliva secreted in the diseased state prevents efficient filling of the capillary. Further, attempts to sweep the oral cavity to collect residual saliva can lead to injury. The pipet method allows for complete collection, and for the same operator, it is remarkably consistent between experiments. However, for unknown reasons, this method shows significant variation between different operators. Therefore, to allow appropriate comparisons, it becomes imperative that the same operator performs all the experiments related to a specific project. Clearly, this represents a major disadvantage for a laboratory.
To overcome these issues, a procedure was developed that combines saliva collection methods used for humans and rodents. The swab method, described below for measuring pilocarpine-induced saliva volume is simple, reproducible, not influenced by the operator, and can be performed repeatedly in the same animal. In addition, it allows collecting the saliva for subsequent analyses of proteins, immunoglobulins, or other biomolecules.

<table><tbody><tr><td>&lt;strong&gt;Chemicals for saliva collection&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Pilocarpine hydrochloride</td><td>Alfa Aesar, Tewksbury, MA, USA</td><td>B21410</td><td>Dilute in sterile isotonic saline. Store single use aliquots of 100X stock at -80&lt;sup&gt;o&lt;/sup&gt;C</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Anesthesia Mix solution&lt;/strong&gt;</td><td></td><td></td><td>Mix 1 mL ketamine hydrochloride + 0.5 mL xylazine + 8.5 mL sterile isotonic saline in a sterile vial. Can be used for 3 months.</td></tr><tr><td>Zetamine (Ketamine hydrochloride)</td><td>Vet One, Boise, Idaho, USA</td><td>C3N VT1</td><td>Stock is 100 mg/mL</td></tr><tr><td>Anased (Xylazine)</td><td>Med-Vet International, Mettawa, IL, USA</td><td>RXANASED-20</td><td>Stock is 20 mg/mL</td></tr><tr><td>Isotonic sterile saline</td><td>Vet One, Boise, Idaho, USA</td><td>501032</td><td>Used as diluent</td></tr><tr><td>Artificial tears (lubricant ophthalmic ointment)</td><td>Henry Schien, Dublin, OH, USA</td><td>48272</td><td>Used to prevent eyes from drying during the procedure</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Materials for saliva collection&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>SalivaBio Children&#039;s swab</td><td>Salimetrics, LLC Carlsbad, CA, USA</td><td>N/A</td><td>Individually wrapped swabs</td></tr><tr><td>50 mL polypropylene tubes</td><td>VWR, Radnor, PA, USA</td><td>89004-364</td><td>Cut off the bottom 1 cm of the tube. Make sure that the cut edge is smooth.</td></tr><tr><td>Microcentrifuge tubes 0.6 mL</td><td>VWR, Radnor, PA, USA</td><td>87003-290</td><td>Make holes in the bottom of tube with heated 18 gauge needles</td></tr><tr><td>Microcentrifuge tubes 2 mL</td><td>VWR, Radnor, PA, USA</td><td>87003-298</td><td></td></tr><tr><td>Insulin Syringes with permanently attached needles</td><td>Becton Dickinson and Company, Franklin Lakes, NJ, USA</td><td>324702</td><td></td></tr><tr><td>18 gauge regular bevel needles</td><td>Becton Dickinson and Company, Franklin Lakes, NJ, USA</td><td>305195</td><td></td></tr><tr><td>Sterile scalpel blade #11</td><td>Integra York Inc PA, USA</td><td>4-311</td><td></td></tr><tr><td>Microdissecting forceps</td><td>Roboz, Gaithersburg, MD, USA</td><td>RS-5139</td><td>Serrated angular 0.8 mm tip, 4&quot; length</td></tr><tr><td>Label tape</td><td>Santa Cruz Biotechnology, Dallas, TX, USA</td><td>sc-224487</td><td></td></tr><tr><td>3 channel timer</td><td>Amazon.com, Seattle, WA, USA</td><td>B06W2KCYVN</td><td></td></tr><tr><td>Analytical Balance</td><td>Mettler Toledo, Columbus OH, USA</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Chemicals/ reagents for inducing salivary dysfunction&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>LPS</td><td>Invivogen, San Deigo, CA, USA</td><td>tlrl-b5lps</td><td>Dissolve in endotoxin free water, and store stock solution at 5 mg/mL . dilute in sterile HBSS 100 ug/mL - inject 100 uL/ moue ip</td></tr><tr><td>Imject Alum adjuvant</td><td>Thermo Scientific</td><td>77161</td><td>Dilute 1:1 in sterile saline. Inject intraperitoneally 0.1 mL/mouse</td></tr><tr><td>Rabbit anti-Ro52 antiserum</td><td>Generated in lab</td><td></td><td>Immunization of rabbits with recombinant mouse Ro52</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Chemicals/ Kits for saliva analyses&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Salivary lysozyme estimation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>EnzChek Lysozyme Assay Kit</td><td>Molecular Probes, Eugene, OR, USA</td><td>E-22013</td><td>Used as per manufacturer&#039;s instructions</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Salivary IgA estimation by sandwich ELISA&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mouse IgA</td><td>Southern Biotech, Birmingham, AL, USA</td><td>0106-01</td><td>Standards for sandwich ELISA - range 30 ng/mL to 0.5 ng/mL</td></tr><tr><td>Goat anti- mouse IgA unlabeled</td><td>Southern Biotech, Birmingham, AL, USA</td><td>1040-01</td><td>Coat at 1 ug/mL in bicarbonate buffer</td></tr><tr><td>Goat anti- mouse IgA HRP</td><td>Southern Biotech, Birmingham, AL, USA</td><td>1040-05</td><td>Detection antibody used at 1:4000 dilution</td></tr><tr><td>TMB substrate</td><td>Becton Dickinson and Company, Franklin Lakes, NJ, USA</td><td>555214</td><td>Used as per manufacturer&#039;s instructions</td></tr><tr><td>Immulon 4HBX Microtiter 96 well plates</td><td>Thermo Scientific, Rochester, NY, USA</td><td>3855</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Salivary protein estimation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Bio-Rad Protein Assay Dye Reagent Concentrate</td><td>BioRad, Hercules, CA, USA</td><td>5000006</td><td>For protein estimation as per manufacturer&#039;s instructions</td></tr></tbody></table>

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.

In the experimental mouse model systems, the ability to produce saliva in response to pilocarpine stimulation is used as an indicator of salivary gland function. Saliva production was measured in 11-week-old C57BL/6 female mice by the swab method. In Figure 1A, the results are expressed as the amount of saliva (mg) collected following increasing doses of pilocarpine. At the dose of 1 mg/kg body weight, some of the mice started exhibiting distress (involuntary shaking and shivering). Hence higher doses of pilocarpine, beyond 1 mg/kg body weight were not tested in this study. In Figure 1B, the results are represented as the ratio of saliva weight (mg) to the mouse body weight (g). Collectively, these data show a good dose response relationship between pilocarpine amount and saliva production. The volume of saliva recovered from the swab was also measured. As shown in Figure 1C, there is a significant concordance between saliva weight and volume where 1 mg of saliva equals 0.001 mL.
The results obtained with the swab method are similar to those obtained with the pipette collection method. Figure 2A shows that the mean amount of saliva collected by the swab method and the pipet method is very similar and the differences are statistically not significant. Figure 2B, 2C and 2D show that the swab method did not impact estimation of biomolecules in saliva. In fact, in comparison with the pipet method, the swab method showed an overall higher trend in the mean levels of total salivary protein (Figure 2C), salivary lysozyme activity (Figure 2D) and salivary IgA (Figure 2E). However, these differences were statistically not significant.
The saliva collection method described here is able to detect differences in the amount of saliva produced by different mouse strains (Figure 3) and salivary gland hypofunction induced by different disease conditions (Figure 4). Figure 3A shows results of pilocarpine induced saliva production from 10-12-week-old female C57BL/6, BALB/c, DBA1/J and 129S6 mice. The 129S6 mice produced the lowest amount of saliva compared to the other strains. It should be noted that the differences in the body weights of these mice were not significantly different (Figure 3B). Next, the swab method was used to detect salivary gland hypofunction, and two examples are shown in Figure 4. Figure 4A shows a representative result of salivary gland hypofunction induced by the activation of innate immunity following injection of lipopolysaccharide (LPS) (10 &#181;g/mouse, intraperitoneally). Control mice were injected with saline11. LPS-treated mice show a significant drop in saliva compared to controls. Passive transfer of antibodies reactive with Sj&#246;gren&#39;s syndrome antigen A/Ro52 induces salivary gland hypofunction, and this model mimics certain aspects of Sj&#246;gren&#39;s syndrome9. As shown in Figure 4B, mice injected with adjuvant alum plus anti-Ro52 antibodies together had significantly lower saliva production compared to untreated mice or mice treated only with alum.
The swab method is simple to implement and operator independent. Figure 5 shows saliva production from 10-13-week-old female C57BL/6 mice, in experiments performed at 6 different time points by 2 different operators. Operator I started with only 2 months of mouse handling experience while Operator II had 6 months of prior mouse handling experience, but was only introduced to the saliva collection method for one week. These data were generated in experiments carried out almost one year apart, using 0.375 mg/kg pilocarpine dose. The saliva ratios obtained by both operators show a range of readings (1.84 to 5.87). Operator I&#39;s experiments spanned a period of 10 weeks while Operator II&#39;s experiments were carried out over 3 consecutive days almost one year later (Figure 5A). Notably, the saliva ratios obtained over time were not significantly different (p=0.064; Kruskal-Wallis test). To further compare inter-operator variations, the ratios of saliva weight per g body weight from 3 experiments for each operator were pooled and are shown in Figure 5B. Saliva ratios obtained by Operator I (mean + SEM; 4.45 + 0.152, n=38) are not significantly different from Operator 2 (mean + SEM; 4.21 + 0.169; n=25; p=0.296 by Mann Whitney test).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57203/57203fig1.jpg" /> 
Figure 1: Saliva production is dependent on pilocarpine dose. C57BL6/J mice (11-week-old females, 5 per group) were injected with different doses of pilocarpine (0.25 mg/kg, 0.5 mg/kg and 1.0 mg/kg body weight) and saliva production was measured for 15 min. (A) Results are represented as mg saliva amount (mean &#177; SEM). (B) Data are represented as mean saliva ratio (mg saliva per g mouse body weight). (C) Saliva from 11-week-old female C57BL/6 mice (n=5) was collected by the swab method. Pilocarpine was used at a dose of 0.375 mg/kg body weight. The wet swabs were weighed to measure the amount of saliva produced in mg. The saliva in the swabs was then recovered by centrifugation, and the volumes of the recovered saliva were measured. The significant agreement is seen between the saliva weight in mg and saliva volume in &#181;L. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57203/57203fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57203/57203fig2.jpg" /> 
Figure 2: The swab method does not impact the analysis of biomolecules in saliva. Saliva from 2 groups of 11-week-old female C57BL/6 mice, (n=5 mice per group), was collected either by the swab method or by the micropipet method. The differences in mean volumes of saliva (A), mean amounts of protein (B), mean amounts of lysozyme activity (C), and mean amounts of IgA between the 2 groups are statistically not significant. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57203/57203fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57203/57203fig3.jpg" /> 
Figure 3: The swab method of saliva collection detects differences in base line saliva production by different strains of mice. (A) Saliva from female (10-12-week-old) mice were collected for 15 min by using a pilocarpine dose of 0.375 mg/kg body weight. (B) The mean body weights between the groups of mice were not significantly different. Statistical significance was analyzed by the Kruskal-Wallis test, followed by Dunn&#39;s multiple comparisons test. A p&#60;0.05 was considered significant. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57203/57203fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57203/57203fig4.jpg" /> 
Figure 4: Swab method detects salivary gland hypofunction. (A) (B6 X A/J) F1 female mice were injected with either LPS solution (0.1 mg/mL, 0.1 mL per mouse, intraperitoneally) or saline, and saliva was measured 26 h later. A representative experiment shows a significant (p=0.0079) drop (60%) in saliva production in mice treated with LPS. (B) Salivary gland hypofunction was measured in an experimental mouse model system for Sj&#246;gren&#39;s syndrome. The previously published passive transfer model for induction of glandular dysfunction was used4 with few modifications. Female C57BL/6 mice (10-12 weeks old) were injected with alum adjuvant. On days 14 and 20 mice were injected with 0.05 mL of rabbit anti-Ro52 serum and saliva production was measured after 24 h. Note a significant (p=0.0003) drop (50%) in saliva production in mice treated with alum and anti-Ro52 serum. Statistical significance was analyzed by the Kruskal-Wallis test, followed by Dunn&#39;s multiple comparisons test. A p&#60;0.05 was considered significant. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57203/57203fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57203/57203fig5.jpg" /> 
Figure 5: The swab method yields reproducible results over time (A) and between operators (B). The swab method shows comparable results between two operators with different lengths of mouse handling experience in experiments carried out over one year. Baseline saliva production in C57BL/6 female mice (10-13 weeks of age) was measured by each operator. Results are expressed as weight of saliva produced (mg/g body weight). Dates of saliva collection are on the X-axis and the results from data collected over time are shown (A). Each data point represents one mouse, and the number of mice analyzed at each time is shown in parenthesis. Baseline saliva ratios (mean + SEM) pooled from 3 experiments are shown (B), and are not significantly different between operators. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57203/57203fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Method for the Measurement of Salivary Gland Function in Mice at JoVE.com

A Method for the Measurement of Salivary Gland Function in Mice

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 ...

a-method-for-the-measurement-of-salivary-gland-function-in-mice

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

18.6K Views.  Oklahoma Medical Research Foundation. 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.

Video: A Method for the Measurement of Salivary Gland Function in Mice

Cannulation of the Mouse Submandibular Salivary Gland via the Wharton's Duct

Severe salivary gland hypofunction is frequently found in patients with Sj&ouml;gren's syndrome and those who receiving therapeutic 
irradiation in their head and neck regions for cancer treatment. Both groups of patients experience symptoms such as xerostomia (dry mouth), dysphagia 
(impaired chewing and swallowing), severe dental caries, altered taste, oro-pharyngeal infections (candidiasis), mucositis, pain and discomfort.
One innovative approach of regenerative medicine for the treatment of salivary gland hypo-function is speculated in RS Redman, E Mezey 
et al. 2009: stem cells can be directly deposited by cannulation into the gland as a potent method in reviving the functions of the impaired organ. Presumably, 
the migrated foreign stem cells will differentiate into glandular cells to function as part of the host salivary gland. Also, this cannulation technique is an 
expedient and effective delivery method for clinical gene transfer application.
Here we illustrate the steps involved in performing the cannulation procedure on the mouse submandibular salivary gland via the Wharton's duct 
(Fig 1). C3H mice (Charles River, Montreal, QC, Canada) are used for this experiment, which have been kept under clean conventional conditions at 
the McGill University animal resource center. All experiments have been approved by the University Animal Care Committee and were in accordance with the 
guidelines of the Canadian Council on Animal Care.
For this experiment, a trypan blue solution is infused into the gland through the opening of the Wharton's duct using a 
insulin syringe with a 29-gauge needle encased inside a polyethylene tube. Subsequently, the mouse is dissected to show that the infusions migrated 
into the gland successfully.

A protocol for the cannulation of the mouse submandibular salivary gland via the Wharton's duct is described. For this experiment, the trypan blue solution is used as a dyer to demonstrate how this technique effectively delivers infusions into the targeted gland, and to suggest the reliability of this new approach as a potential clinical drug/cell therapy for the regeneration of salivary glands.

Severe salivary gland hypofunction is frequently found in patients with Sj&ouml;gren's syndrome and those who receiving therapeutic 
irradiation in their ...

Medicine

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Biology

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

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 ...

Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation

Hyposalivation is commonly observed in the autoimmune reaction of Sj&#246;gren's syndrome or following radiation injury to the major salivary glands. In these cases, questions remain regarding disease pathogenesis and effective interventions. An optimized technique that allows functional assessment of the salivary glands is invaluable for investigating exocrine gland biology, dysfunction, and therapeutics. Here, we present a step by step approach to performing pilocarpine stimulated saliva secretion, including tracheostomy and the dissection of the three major murine salivary glands. We also detail the appropriate murine head and neck anatomy accessed during these techniques. This approach is scalable, allowing for multiple mice to be processed simultaneously, thus improving the efficiency of the work flow. We aim to improve the reproducibility of these methods, each of which has further applications within the field. In addition to saliva collection, we discuss metrics for quantifying and normalizing functional capacity of these tissues. Representative data are included from submandibular glands with depressed salivary gland function 2 weeks following fractionated radiation (4 doses of 6.85 Gy).

We present a detailed approach to performing saliva collection, including murine tracheostomy and the isolation of three major salivary glands.

Hyposalivation is commonly observed in the autoimmune reaction of Sj&#246;gren's syndrome or following radiation injury to the major salivary glands. In ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

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 ...

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection

Rice stripe virus (RSV), which causes significant economic loss of agriculture in East Asia, entirely depends on insect vectors for its effective transmission among host rice. Laodelphax striatellus (small brown planthopper, SBPH) is the primary insect vector that horizontally transmits RSV while sucking sap from the phloem. Saliva plays a significant role in insects' feeding behavior. A convenient method that will be useful for research on insects' saliva with piercing-sucking feeding behavior is described here. In this method, insects were allowed to feed on an artificial diet sandwiched between two stretched paraffin film layers. The diet containing the saliva was collected each day, filtered, and concentrated for further analysis. Finally, the quality of collected saliva was examined by protein staining and immunoblotting. This method was exemplified by detecting the presence of RSV and a mucin-like protein in the saliva of SBPH. These artificial feeding and saliva collection method will lay a foundation for further research on factors in insect saliva related to feeding behavior and virus transmission.

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection

The present protocol describes a method to collect sufficient saliva from piercing-sucking insects using an artificial medium. This is a convenient method for collecting insect saliva and studying salivary function on insect feeding behavior and vector-borne virus transmission.

Rice stripe virus (RSV), which causes significant economic loss of agriculture in East Asia, entirely depends on insect vectors for its effective ...

Noninvasive Real-Time Whole-Body Plethysmography for Cough Sensitivity in Mice

Methods for Detecting Cough and Airway Inflammation in Mice

Chronic cough, which lasts for more than 8 weeks, is one of the most common complaints requiring medical attention, and patients suffer from a huge socioeconomic burden and a marked decrement in quality of life. Animal models can mimic the complex pathophysiology of the cough and are important tools for cough research. The detection of cough sensitivity and airway inflammation is of great significance for studying the complex pathological mechanism of cough. This article describes the measurement of cough using a noninvasive and real-time whole-body plethysmography (WBP) system and the normative procedures for harvesting tissue samples (including blood, lung, spleen, and trachea) of mice. It introduces some methods to assess airway inflammation, including pathological changes in hematoxylin and eosin (HE)-stained lung and trachea sections, the total protein concentration, the uric acid concentration, and the lactate dehydrogenase (LDH) activity in the supernatant of bronchoalveolar lavage fluid (BALF), and the leukocytes and differential cell counts of BALF. These methods are reproducible and serve as valuable tools to study the complex pathophysiology of cough.

Here, we describe the measurement of cough using a noninvasive and real-time whole-body plethysmography (WBP) system and the normative procedures for harvesting tissue samples of mice and introduce some methods to assess airway inflammation.

Chronic cough, which lasts for more than 8 weeks, is one of the most common complaints requiring medical attention, and patients suffer from a huge ...

A Method for the Measurement of Salivary Gland Function in Mice

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Przeglądaj więcej filmów

Cannulation of the Mouse Submandibular Salivary Gland via the Wharton's Duct

Isolation of Mouse Salivary Gland Stem Cells

Evaluation of Mammary Gland Development and Function in Mouse Models

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

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

Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

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

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection

Methods for Detecting Cough and Airway Inflammation in Mice