We would like to thank Ms. Amy Iverson in the Department of Infectious Diseases at St. Jude Children's Research Hospital for technical training on influenza virus culture and infections and Dr. Stacie Woolard in the Department of Flow Cytometry at St. Jude Children's Research Hospital for help in setting up the 14 color antibody cocktail for analysis. We would also like to thank Arthur Covington and Shelby Patrick of St. Jude for animal husbandry and care. These studies were supported by the National Institute of Allergy and Infectious Diseases, the National Institutes of Health, under contract number HHSN26620070005C, Centers of Excellence in Influenza Research and Surveillance (CEIRS), N 01 7005C (JAM) and Le Bonheur Junior Faculty Grant (Samarasinghe).

Ethics Statement. All experiments were approved by the Institutional Animal Use and Care Committee at St. Jude Children&#39;s Research Hospital.
Note: All described experiments in this protocol must be conducted under BSL-2 and ABSL-2 conditions since Aspergillus fumigatus and influenza A virus are BSL-2 agents.
1. Modeling Allergic Asthma in Mice (Figure 1)
<ol>
	<li>Fungal sensitization
	<ol>
		<li>Re-constitute A. fumigatus antigen in sterile PBS at 1 mg protein/mL. Aliquot and store at -20&#176;C.</li>
		<li>House animals in a micro-isolator cages with autoclaved paper bedding under specific pathogen-free conditions with ad libitum food and water in a 12 hour light:dark cycle. Obtain C57BL/6J female mice at six weeks of age and acclimatize for one week. Start mice on the asthma protocol at seven weeks of age.</li>
		<li>Dilute A. fumigatus antigen to 20 &#181;g/mL in sterile PBS and add dropwise to an equal volume of Alum while stirring. Allow antigen to be adsorbed for 30 mins at room temperature with constant stirring.</li>
		<li>Fill 1 mL luer-lock syringes with the mixture and deliver 0.1 mL intraperitoneally and 0.1 mL subcutaneously between the ears to each mouse. Administer PBS and Alum as a mock-sensitization to mice that serve as na&#239;ve controls.</li>
		<li>Two weeks after the initial sensitization, dilute A. fumigatus antigen to a concentration of 200 &#181;g/mL in sterile PBS and deliver 20 &#181;L intranasally to each mouse. Repeat once weekly for a total of three intranasal administrations. Administer PBS as a mock-sensitization to mice that serve as na&#239;ve controls.</li>
	</ol>
	</li>
	<li>Fungal challenge
	<ol>
		<li>Prepare Sabouraud dextrose agar at 65 g/L per manufacturer&#39;s guidelines and sterilize in an autoclave at 121&#176;C for 15 mins. Place in a water bath set at 50&#176;C. to temper.</li>
		<li>Once tempered, aseptically add 10 mL to 25 cm2 canted neck, vented cap cell culture flasks. Lay the flasks flat and allow agar to set. Once set, recap flasks and store at 4&#176;C until use.</li>
		<li>Re-suspend the A. fumigatus culture aseptically by adding 5 mL of sterile PBS. Store overnight at 4&#176;C. Prepare 60 &#181;L aliquots for use and store at 4&#176;C.</li>
		<li>Eight days before the final intranasal administration of A. fumigatus antigen, culture A. fumigatus on Sabouraud dextrose agar for the fungal challenge. Add one well vortexed 60 &#181;L aliquot of fungus in each agar flask and spread using a disposable inoculating loop. Incubate flasks at 37&#176;C for 8 days.</li>
		<li>Set up the inhalation challenge apparatus in a class II biological safety cabinet as described in detail by Schuh and Hoselton.7 Create holes in the fungal culture flasks, one on the rear top and one on the bottom of the flask immediately above the culture by using a cork-borer.</li>
		<li>Attach the fungal culture flask to the regulated medical grade air supply through the bottom hole and to the inhalation chamber through the top hole. Cover the inhalation ports with laboratory tape.</li>
		<li>Run medical grade air over the culture at 2 psi for 10 mins to coat the chamber. Prepare sterile anesthetic cocktail with 75 mg/kg ketamine and 25 mg/kg xylazine.</li>
		<li>Inject the drug cocktail to three mice intraperitoneally and determine anesthesia by toe pinch reflex and lack of movement. Apply a drop of lubricating ointment in each eye to prevent drying out.</li>
		<li>Turn off the air, and replace the fungal flask with a new one and open the ports of the inhalation chamber.</li>
		<li>Place the anesthetized mice supine with their noses in the ports. Turn on the air over the culture and allow the animals to inhale the liberated fungal conidia for 10 mins.</li>
		<li>Place each animal supine in a new cage and place the cage on a heating pad. Monitor animals for recovery from anesthesia marked by turning over and movement around the cage.</li>
		<li>Repeat procedure after 14 days. Anesthetize animals that serve as na&#239;ve controls and place in a new cage.</li>
	</ol>
	</li>
</ol>
2. Modeling Influenza in Mice
NOTE: Adjust the age, gender, and strain of mice and the dose of the virus according to the experimental goals.
<ol>
	<li>Obtain female C57BL/6J mice at 14 or 17 weeks of age and acclimatize to the facility in micro-isolator cages with autoclaved paper bedding and ad libitum food and water in a 12 hour light:dark cycle. Number animals in each cage by tail mark or ear notch. One week later, weigh each mouse and record weight with date and time.</li>
	<li>Thaw a stock of A/CA/04/2009 virus and dilute in sterile PBS at log104.305 TCID50/mL in a glass vial. Anesthetize mice with 2.5% isoflurane with oxygen in an anesthesia chamber within the class II biological safety cabinet.</li>
	<li>Deliver 50 &#181;L of virus intranasally to each mouse for a final dose of 1000 TCID50 pH1N1 per mouse. Hold mouse supine until conscious and place in new micro-isolator cage. Administer PBS under anesthesia to na&#239;ve controls.</li>
	<li>Record animal weights every 24 hours until experimental endpoints are reached. 
	NOTE: It is good practice to use the same stock virus tube(s) to infect all animals in the study and not use previously thawed tubes as the viral titer may decrease during repeated freeze-thaw cycles.</li>
</ol>
3. Modeling Asthma and Influenza Co-morbidity in Mice (Figures 2 and 3)
<ol>
	<li>To mimic acute asthma and influenza, infect animals one week after the second fungal challenge with pH1N1 as noted in section 2. To mimic chronic asthma and influenza, infect animals 4 weeks after the second fungal challenge.</li>
</ol>
4. Pulmonary function test
<ol>
	<li>Weigh and record each mouse weight.</li>
	<li>Perform pulmonary function test on each animal. Note: Of the various equipment available to measure pulmonary function in rodents, we have opted to use the flexiVent FX1 module and flexiware software as previously described.22
	<ol>
		<li>Open the software and go to &#34;Study Definition and Planning.&#34; Click on &#34;Create a new study&#34; to launch the Study Definition Wizard.</li>
		<li>Follow the Wizard to input details of the study: Title and Status, Objective and Hypothesis; Protocol; Subject (animal) Groups.</li>
		<li>Create subjects by clicking on the mouse icon and add all necessary information on the animal including the Group Membership which will assign subjects to Subject Groups.</li>
		<li>Verify all information and confirm to close the wizard.</li>
	</ol>
	</li>
	<li>After concluding the &#34;Study Definition and Planning&#34; session, open the &#34;Experimentation Session&#34; in the software. Follow the steps of the pop-up wizard to set-up the instrument.
	<ol>
		<li>Select the Study (see 4.2.1) and the Experiment Template which depends on the instrument.</li>
		<li>Add details as deemed necessary to Session Properties. The instrument will then be connected to the software.</li>
		<li>Select the instrument module attached to the software when promoted and click OK.</li>
		<li>Select a subject in the Study to Site 1 and enter the current weight of the animal when prompted.</li>
		<li>Add 0.5 mL of saline to the finemist nebulizer and click OK when prompted to Prime the nebulizer (the generated mist will be visible). Attach the nebulizer to the instrument adaptor when complete.</li>
		<li>Perform Channel Calibrations when prompted. Calibrate the Cylinder Pressure and Airway Opening Pressure following the software wizard using a manometer to apply known pressures.</li>
		<li>At the conclusion of Channel Calibrations, the Tube Calibration Wizard will be initiated; attach an 18 g cannula to the Y-tubing adaptor and select the desired perturbations. Perform closed and open calibrations and verify that Calibration Results are void of errors.</li>
		<li>Once the calibrations are complete, select whether to start recording continuous data when prompted. Click &#34;Yes&#34; to start the default ventilation pattern. The instrument is ready to gather data.</li>
	</ol>
	</li>
	<li>Prepare a stock acetyl-&#946;-methylcholine (A&#946;M) at 125 mg/mL in sterile saline; freeze aliquots at -20&#176;C until use. Dilute the frozen stock of A&#946;M to the desired doses in sterile saline (3.125 mg/mL, 6.25 mg/mL, 12.5 mg/mL, and 25 mg/mL).</li>
	<li>Prepare anesthetics in sterile PBS before procedure. Administer 10 mg/kg body weight xylazine intraperitoneally and place mouse its own cage to rest for 5 min. Administer 70 mg/kg body weight pentobarbital intraperitoneally and allow mouse to rest for 5 min.</li>
	<li>Intubate mouse as previously described.23
	<ol>
		<li>Briefly, place the anesthetized mouse supine on a slanted surgical board and fix in place with plastic tape over the forelimbs and tail.</li>
		<li>Make a small incision between the upper thorax and lower jaw. Separate the salivary glands and thyroid gland to expose the trachea. Gently pass curved surgical forceps underneath the trachea and pull a piece of thread underneath.</li>
		<li>Perform tracheostomy between two tracheal rings with sharp scissors taking care not to cut through the trachea.</li>
		<li>Insert the 18 g cannula that was used to calibrate the instrument. Fasten the cannula to the trachea with thread and attach the mouse to the Y-tubing of the instrument adaptor with the cannula.</li>
	</ol>
	</li>
	<li>Perform a deep inflation to recruit the airways and help the animal succumb to the ventilator. Select the &#34;mouse inhaled dose response v7.0&#34; script with default ventilation at 150 breaths/minute.</li>
	<li>Add saline followed by each incremental dose of A&#946;M to the nebulizer when prompted to perform a dose response curve.</li>
	<li>At the conclusion of the data acquisition after the highest A&#946;M dose, disconnect the mouse from the ventilator and humanely euthanize the animal by performing a cervical dislocation. Proceed to tissue harvest.</li>
	<li>Add saline to the nebulizer and activate the nebulizer to remove any remnant high dose A&#946;M. Change the subject in the software program and proceed with next mouse beginning with a tube calibration (step 4.1-4.3).</li>
</ol>
5. Tissue harvest
<ol>
	<li>Spray euthanized mouse with 70% ethanol and open the thoracic cavity. Open diaphragm and perform bronchoalveolar lavage (BAL) with 1 mL of sterile PBS injected through the 18 g cannula and gently draw out repeating once for a total of 2 mL.24 Place the sample in ice.</li>
	<li>Harvest the cardiac and diaphragmatic lobes of the right lung for RNA work, and azygous and apical lobes for protein work, place in separate 5 mL snap-cap tubes and snap freeze in liquid nitrogen.</li>
	<li>Harvest the left lobe using blunt forceps and scissors taking care not to damage/slice through the lobe. Fix the lung ex vivo with 10% normal buffered formalin with a 30 g needle. Store in 10% normal buffered formalin until histological analyses. 
	NOTE: When animals are taken through the model for determination of viral burden in the lungs, harvest whole lungs after euthanasia and snap freeze in liquid nitrogen.</li>
</ol>
6. Tissue processing for Flow Cytometry
<ol>
	<li>Centrifuge the BAL contents at 600 g for 10 mins at 4&#176;C and save the BAL fluid for protein assays at -80&#176;C until use.</li>
	<li>Perform red blood cell lysis if necessary noting that cells are more susceptible to death after viral infection. Perform red cell lysis by adding 8 parts cold distilled water and pulse-vortexing for 30 seconds followed by a one-minute incubation at room temperature. Add one part 10 PBS, mix to neutralize the water and centrifuge as above.</li>
	<li>Re-suspend the BAL cells in 200 &#181;L of staining media (sterile PBS with 5% FBS) and enumerate cells by trypan blue dye exclusion assay to determine the cell concentration.</li>
	<li>Cytospin 50,000 cells as follows:
	<ol>
		<li>Transfer the calculated volume of cells into a tube and bring volume up to 100 &#181;L with sterile PBS. Assemble the labeled slide and cytofunnel in the metal clip and place in the centrifuge.</li>
		<li>Add the diluted cells into the cytofunnel and spin at 55 g for 5 minutes to affix the cells to the slides.</li>
		<li>Perform differential staining by sequentially dipping the slides 20 times each in methanol, xanthene, and thiazine followed by distilled water and air-drying.</li>
	</ol>
	</li>
	<li>Add 1 &#181;L of human gamma globulin for every 10,000 cells in the cell suspension in step 6.3 and incubate in ice for 30 mins. Adjust volume to 1 mL with staining media and centrifuge at 600 g for 10 mins at 4&#176;C and aspirate supernatant. Re-suspend cells in 100 &#181;L of staining media and prepare four pooled samples with 10 &#181;L from each sample. Adjust volumes to 200 &#181;L and centrifuge as above.</li>
	<li>Re-suspend cells in 50 &#181;L of antibody cocktail containing fluorochrome-conjugated antibodies diluted in staining media (see table of materials). Re-suspend one of the pooled samples in 50 &#181;L of staining media, one pooled sample in 50 &#181;L of isotype control antibodies, and the remaining pooled samples with diluted antibodies individually. Incubate samples in ice protected from light for 30 mins. 
	NOTE: The use of antibodies will be based on research interests and should be optimized previously for the experiment. Each antibody used should be used individually in a pooled sample for instrument set up. The use of bone marrow cells or splenocytes for single color controls may be necessary if the cell yield is low and is acceptable if fluorochrome-conjugated antibodies are made against an abundant marker such as B220.</li>
	<li>Add 450 &#181;L of staining media and centrifuge at 600 g for 10 mins at 4&#176;C. Aspirate supernatant and repeat.</li>
	<li>Aspirate supernatant and re-suspend cells in 50 &#181;L of freshly prepared stabilizing fixative by gentle pipetting. Add 200 &#181;L more of stabilizing fixative and strain through a 40 &#181;m nylon mesh into a polystyrene FACS tube for analysis by flow cytometry. 
	NOTE: Whole lungs may be digested to make single cell suspensions and used to analyze cell populations by flow cytometry.</li>
</ol>
7. Tissue Processing for RNA Analysis
<ol>
	<li>Place frozen lung lobes in ice within a fume hood, and perform standard Trizol extraction of RNA according to manufacturer&#39;s recommended protocol. After DNase treatment and purification of RNA, convert 1 &#181;g of RNA to cDNA.</li>
	<li>Using RNA-specific validated primer sets, measure changes in gene expression with quantitative real-time PCR and analyze data using the 2-&#916;&#916;Ct method normalized to the internal housekeeping gene25 validated for lungs.</li>
</ol>
8. Tissue processing for Viral Titration
<ol>
	<li>Place frozen lungs in ice and add 1 mL of sterile PBS and mechanically homogenize tissue until entire tissue is pulsed. Centrifuge samples at 600 g for 10 mins at 4&#176;C and store supernatants in aliquots at -80&#176;C until use.</li>
	<li>Quantify virus using standard tissue culture infectious dose 50 (TCID50) method26 with MDCK.2 cells.</li>
</ol>

<ol>
	<li>Borish, L., Culp, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asthma:+a+syndrome+composed+of+heterogeneous+diseases.">Asthma: a syndrome composed of heterogeneous diseases.</a> Ann Allergy Asthma Immunol. 101, 1-11 (2008).</li><li>Denning, D. W., O'Driscoll, B. R., Hogaboam, C. M., Bowyer, P., Niven, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+link+between+fungi+and+severe+asthma:+a+summary+of+the+evidence.">The link between fungi and severe asthma: a summary of the evidence.</a> Eur Respir. J. 27, 615-626 (2006).</li><li>Wenzel, S., Holgate, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+trap:+It+still+yields+few+answers+in+asthma.">The mouse trap: It still yields few answers in asthma.</a> Am J Respir Crit Care Med. 174, 1173-1178 (2006).</li><li>Hoselton, S. A., Samarasinghe, A. E., Seydel, J. M., Schuh, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+inhalation+model+of+airway+allergic+response+to+inhalation+of+environmental+Aspergillus+fumigatus+conidia+in+sensitized+BALB/c+mice.">An inhalation model of airway allergic response to inhalation of environmental Aspergillus fumigatus conidia in sensitized BALB/c mice.</a> Med Mycol. 48, 1056-1065 (2010).</li><li>Schuh, J. M., Hoselton, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+inhalation+model+of+allergic+fungal+asthma:+Aspergillus+fumigatus-induced+inflammation+and+remodeling+in+allergic+airway+disease.">An inhalation model of allergic fungal asthma: Aspergillus fumigatus-induced inflammation and remodeling in allergic airway disease.</a> Methods Mol Biol. 1032, 173-184 (2013).</li><li>Samarasinghe, A. E., Hoselton, S. A., Schuh, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+absence+of+the+VPAC(2)+receptor+does+not+protect+mice+from+Aspergillus+induced+allergic+asthma.">The absence of the VPAC(2) receptor does not protect mice from Aspergillus induced allergic asthma.</a> Peptides. 31, 1068-1075 (2010).</li><li>Samarasinghe, A. E., Hoselton, S. A., Schuh, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+between+intratracheal+and+inhalation+delivery+of+Aspergillus+fumigatus+conidia+in+the+development+of+fungal+allergic+asthma+in+C57BL/6+mice.">A comparison between intratracheal and inhalation delivery of Aspergillus fumigatus conidia in the development of fungal allergic asthma in C57BL/6 mice.</a> Fungal Biol. 115, 21-29 (2011).</li><li>Taubenberger, J. K., Morens, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza:+the+mother+of+all+pandemics.">Influenza: the mother of all pandemics.</a> Emerg Infect Dis. 12, 15-22 (1918).</li><li>. Pandemic Flu History Available from: <a target="_blank" href="https://www.cdc.gov/flu/">https://www.cdc.gov/flu/</a> (2016)</li><li>Dawood, F. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimated+global+mortality+associated+with+the+first+12+months+of+2009+pandemic+influenza+A+H1N1+virus+circulation:+a+modelling+study.">Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study.</a> Lancet Infect Dis. 12, 687-695 (2012).</li><li>Jain, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hospitalized+patients+with+2009+H1N1+influenza+in+the+United+States,+April-June+2009.">Hospitalized patients with 2009 H1N1 influenza in the United States, April-June 2009.</a> N Engl J Med. 361, 1935-1944 (2009).</li><li>Jhung, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+2009+pandemic+influenza+A+(H1N1)+in+the+United+States.">Epidemiology of 2009 pandemic influenza A (H1N1) in the United States.</a> Clin Infect Dis. 52, S13-S26 (2011).</li><li>Chowell, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiological+characteristics+and+underlying+risk+factors+for+mortality+during+the+autumn+pandemic+wave+in+Mexico.">Epidemiological characteristics and underlying risk factors for mortality during the autumn pandemic wave in Mexico.</a> PLoS One. 7, e41069 (2009).</li><li>Kusznierz, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+features+of+the+hospitalized+patients+with+2009+pandemic+influenza+A+(H1N1)+in+Santa+Fe,+Argentina.">Clinical features of the hospitalized patients with 2009 pandemic influenza A (H1N1) in Santa Fe, Argentina.</a> Influenza Other Respir Viruses. 7, 410-417 (2013).</li><li>Nguyen-Van-Tam, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+factors+for+hospitalisation+and+poor+outcome+with+pandemic+A/H1N1+influenza:+United+Kingdom+first+wave+(May-September+2009).">Risk factors for hospitalisation and poor outcome with pandemic A/H1N1 influenza: United Kingdom first wave (May-September 2009).</a> Thorax. 65, 645-651 (2009).</li><li>Gilca, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+factors+for+hospitalization+and+severe+outcomes+of+2009+pandemic+H1N1+influenza+in+Quebec,+Canada.">Risk factors for hospitalization and severe outcomes of 2009 pandemic H1N1 influenza in Quebec, Canada.</a> Influenza Other Respi Viruses. 5, 247-255 (2011).</li><li>McKenna, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asthma+in+patients+hospitalized+with+pandemic+influenza+A(H1N1)pdm09+virus+infection-United+States,+2009.">Asthma in patients hospitalized with pandemic influenza A(H1N1)pdm09 virus infection-United States, 2009.</a> BMC Infect Dis. 13, 57 (2013).</li><li>Myles, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+between+asthmatics+and+nonasthmatics+hospitalised+with+influenza+A+infection.">Differences between asthmatics and nonasthmatics hospitalised with influenza A infection.</a> Eur Respir J. 41, 824-831 (2013).</li><li>Chang, Y. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+lymphoid+cells+mediate+influenza-induced+airway+hyper-reactivity+independently+of+adaptive+immunity.">Innate lymphoid cells mediate influenza-induced airway hyper-reactivity independently of adaptive immunity.</a> Nat Immunol. 12, 631-638 (2011).</li><li>McGovern, T. K., Robichaud, A., Fereydoonzad, L., Schuessler, T. F., Martin, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+respiratory+system+mechanics+in+mice+using+the+forced+oscillation+technique.">Evaluation of respiratory system mechanics in mice using the forced oscillation technique.</a> J Vis Exp. , e50172 (2013).</li><li>Moldestad, O., Karlsen, P., Molden, S., Storm, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracheotomy+improves+experiment+success+rate+in+mice+during+urethane+anesthesia+and+stereotaxic+surgery.">Tracheotomy improves experiment success rate in mice during urethane anesthesia and stereotaxic surgery.</a> J Neurosci Methods. 176, 57-62 (2009).</li><li>Daubeuf, F., Frossard, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performing+Bronchoalveolar+Lavage+in+the+Mouse.">Performing Bronchoalveolar Lavage in the Mouse.</a> Current Protocols in Mouse Biology. 2, 167-175 (2012).</li><li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2(-Delta+Delta+C(T))+Method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.</a> Methods. 25, 402-408 (2001).</li><li>Carter, J. B., Saunders, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Virology Principles and Applications. , 9-29 (2007).</li><li>Samarasinghe, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immune+profile+associated+with+acute+allergic+asthma+accelerates+clearance+of+influenza+virus.">The immune profile associated with acute allergic asthma accelerates clearance of influenza virus.</a> Immunol Cell Biol. 92, 449-459 (2014).</li><li>Gern, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ABCs+of+rhinoviruses,+wheezing,+and+asthma.">The ABCs of rhinoviruses, wheezing, and asthma.</a> J Virol. 84, 7418-7426 (2010).</li><li>Han, J., Takeda, K., Gelfand, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+RSV+Infection+in+Asthma+Initiation+and+Progression:+Findings+in+a+Mouse+Model.">The Role of RSV Infection in Asthma Initiation and Progression: Findings in a Mouse Model.</a> Pulm Med. 2011, 748038 (2011).</li><li>Hsu, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+thymic+involution+in+C57BL/6J+x+DBA/2J+recombinant-inbred+mice+maps+to+mouse+chromosomes+9+and+10.">Age-related thymic involution in C57BL/6J x DBA/2J recombinant-inbred mice maps to mouse chromosomes 9 and 10.</a> Genes Immun. 4, 402-410 (2003).</li><li>Chang, Y. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+infection+in+suckling+mice+expands+an+NKT+cell+subset+that+protects+against+airway+hyperreactivity.">Influenza infection in suckling mice expands an NKT cell subset that protects against airway hyperreactivity.</a> J Clin Invest. 121, 57-69 (2011).</li><li>Wohlleben, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+A+virus+infection+inhibits+the+efficient+recruitment+of+Th2+cells+into+the+airways+and+the+development+of+airway+eosinophilia.">Influenza A virus infection inhibits the efficient recruitment of Th2 cells into the airways and the development of airway eosinophilia.</a> J Immunol. 170, 4601-4611 (2003).</li><li>Mori, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+respiratory+syncytial+virus+and+influenza+infection+in+a+house-dust-mite-induced+asthma+mouse+model:+consequences+for+steroid+sensitivity.">Differences in respiratory syncytial virus and influenza infection in a house-dust-mite-induced asthma mouse model: consequences for steroid sensitivity.</a> Clin Sci (Lond). 125, 565-574 (2013).</li><li>Myles, P. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predictors+of+clinical+outcome+in+a+national+hospitalised+cohort+across+both+waves+of+the+influenza+A/H1N1+pandemic+2009-2010+in+the+UK.">Predictors of clinical outcome in a national hospitalised cohort across both waves of the influenza A/H1N1 pandemic 2009-2010 in the UK.</a> Thorax. 67, 709-717 (2012).</li><li>van der Zalm, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+frequent+infections+with+human+rhinovirus+in+healthy+young+children:+a+longitudinal+cohort+study.">Highly frequent infections with human rhinovirus in healthy young children: a longitudinal cohort study.</a> J Clin Virol. 52, 317-320 (2011).</li></ol>

The authors have nothing to disclose.

The high incidences of allergic asthma and influenza increase the likelihood that these two diseases would occur simultaneously in the same individual. While some viruses are clearly demonstrated to be causative agents initiating the pathogenesis of asthma,28,29 the impact of influenza virus infections on either the development or exacerbation of asthma is unclear. The association and the importance of studying influenza viruses in the context of allergic disease became evident when asthma was identified as a ...

Commonly referred to as a disease, asthma is a syndrome1 that affects over 235 million of the world's population.2 Allergic asthma is the most prevalent type of asthma and results from exposures to environmental factors such as pollen, house dust mite and cockroach antigens, and fungi.3 Fungal antigens are common sensitizing allergens affecting 25% of persistent asthmatics and can cause life threatening asthma attacks.4 The development of an accurate model of allergic asthma is challenging because mice do not naturally develop asthma and there are differences between mouse and human respiratory systems.5 The selection of the mouse asthma model system is largely dependent on the experimental question since the output may vary with the antigen used. A fungal asthma model using Aspergillus fumigatus conidia used in native form and natural route of entry into the airways of mice, described in detail elsewhere,6-8 has been shown to elicit all the hallmarks of human disease including airways hyperresponsiveness, eosinophilic inflammation, mucus hypersecretion, and airway wall remodeling events.9
Influenza affects millions worldwide every year. Genetic alterations that occur in influenza viruses by antigenic drifts and shifts, and re-assortments in common hosts can result in a novel virus with the potential to cause a pandemic. The influenza pandemic of 1918 (Spanish Flu) claimed over 50 million lives mostly of young adults.10 The first influenza pandemic of the 21st century occurred in 2009. Although the 2009 influenza pandemic was not as severe as that of 1918, approximately 90 million people were infected11 of which a little over 200,000 died.12 Hospitalized patients included those with underlying diseases such as obesity, cardiovascular and metabolic disease, asthma, COPD, and diabetes.13-16 Although asthma was identified as a risk factor for severe influenza morbidity,17,18 asthmatics were less likely to be admitted to the intensive care unit and die compared to patients without asthma.19,20 The reasons for these seemingly counterintuitive findings are unknown. Therefore, a reliable animal model system was necessary to study the intersection of these two diseases. 
Since other respiratory viruses such as rhinovirus and respiratory syncytial virus (RSV) have been shown to induce asthma, existing animal models of asthma and influenza explored whether influenza virus infections induces the development of asthma.21 The airways, lung parenchymal tissue, and the immune responses are different during acute allergic asthma exacerbations and chronic stable asthma. Therefore, models that can explore how influenza virus infections intersect allergic asthma in each situation are necessary to understand the underlying mechanisms of asthma and influenza comorbidities. This protocol describes a mouse model that can recapitulate these two scenarios.

<table border="0" cellpadding="0" cellspacing="0" width="693">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Aspergillus fumigatus antigen</td>
			<td style="width:124px;">Greer Labs</td>
			<td style="width:131px;">XPM3D3A25</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Aspergillus fumigatus</td>
			<td style="width:124px;">ATCC</td>
			<td style="width:131px;">NIH 5233</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Imject Alum</td>
			<td style="width:124px;">Pierce</td>
			<td style="width:131px;">77161</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Difco Sabouraud dextrose agar</td>
			<td style="width:124px;">BD&#160;</td>
			<td style="width:131px;">210950</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Spore-Klenz</td>
			<td style="width:124px;">Fisher Scientific</td>
			<td style="width:131px;">04-324-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Ketamine</td>
			<td style="width:124px;">Hospira</td>
			<td style="width:131px;">0409-2051-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Xylazine</td>
			<td style="width:124px;">Akorn Inc.</td>
			<td style="width:131px;">59399-110-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Isothesia (Isoflurane)</td>
			<td style="width:124px;">Butler Schein</td>
			<td>05260-04-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Euthasol</td>
			<td style="width:124px;">Virbac</td>
			<td style="width:131px;">710101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Acetyl-&#946;-methylcholine</td>
			<td style="width:124px;">Sigma Aldrich</td>
			<td style="width:131px;">A2251</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">flexiVent FX1</td>
			<td style="width:124px;">SCIREQ</td>
			<td style="width:131px;">FV-FXM1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Nebulizer (standard mist)</td>
			<td style="width:124px;">SCIREQ</td>
			<td style="width:131px;">ANP-1100</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:227px;">flexiWare Software</td>
			<td style="width:124px;">SCIREQ</td>
			<td style="width:131px;">version 7.0.1 rel B</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:227px;">Quik-Dip 3 Part Differential Stain</td>
			<td style="width:124px;">Mercedes Medical</td>
			<td style="width:131px;">MER 1002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Human gamma globulin</td>
			<td style="width:124px;">Sigma Aldrich</td>
			<td style="width:131px;">G4386</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Rat anti-mouse CD3e-PE-Cy7</td>
			<td style="width:124px;">BD Biosciences</td>
			<td style="width:131px;">552774</td>
			<td>Clone: 145-2C11; Diluted 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Hamster anti-mouse CD8&#945;-FITC</td>
			<td style="width:124px;">BD Biosciences</td>
			<td style="width:131px;">553031</td>
			<td>Clone: 53-6.7, Diluted 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">FITC Rat IgG2a</td>
			<td style="width:124px;">BD Biosciences</td>
			<td style="width:131px;">553929</td>
			<td>Diluted 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">PE-Cy7 Armenian Hamster IgG</td>
			<td style="width:124px;">BD Biosciences</td>
			<td style="width:131px;">400922</td>
			<td>Clone: HTK888; Diluted 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Stabilizing fixative</td>
			<td style="width:124px;">BD Biosciences</td>
			<td style="width:131px;">338036</td>
			<td>Freshly prepare&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">MDCK.2</td>
			<td style="width:124px;">ATCC</td>
			<td style="width:131px;">CRL-2936</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">TRIzol Reagent</td>
			<td style="width:124px;">Life technologies</td>
			<td style="width:131px;">15596-026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Chloroform</td>
			<td style="width:124px;">IBI Scientific</td>
			<td style="width:131px;">IB05040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">iScript</td>
			<td style="width:124px;">Bio-Rad</td>
			<td style="width:131px;">170-8891</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">QuantiTect Primer Assays: HPRT1</td>
			<td style="width:124px;">Qiagen</td>
			<td style="width:131px;">QT00166768</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling asthma and influenza co-morbidity in c57bl/6j mice

Allergic asthma and influenza are both diseases of the pulmonary system that affect millions worldwide. During the 2009 influenza pandemic, asthma was identified as an underlying disease in hospitalized patients, although reasons for this increased susceptibility were unknown. Animal models are necessary to explore mechanisms of disease pathogenesis. However, models that could be used to study influenza virus infections in existing asthma are lacking. This protocol describes the development of mouse model systems of asthma and influenza comorbidity using an Aspergillus fumigatus-induced asthma model and the 2009 pandemic H1N1 influenza strain A/CA/04/2009. The host responses that occur in acute and chronic asthma can be explored by changing the timing of the influenza virus infection. These models suggest that the allergic host response to influenza virus depends on the state of the allergic airways and level of allergic inflammation.

The development and characterization of mouse models that recapitulated patient subsets with asthma and influenza is described. Such models are important in understanding host-pathogen interactions that occur during influenza virus infections in hosts that have underlying disease in the pulmonary system which alters the immune response to the invading virus.
Fungal asthma model
<p class="jove_content" fo:keep-togeth...

Asthma and influenza are diseases affecting the pulmonary system that affects millions worldwide. The aim of this study was to develop a mouse model of asthma and influenza comorbidity to study the intersection of these two diseases in the same host.

Modeling Asthma and Influenza Co-morbidity in C57BL/6J Mice

Allergic asthma and influenza are both diseases of the pulmonary system that affect millions worldwide. During the 2009 influenza pandemic, asthma was ...

modeling-asthma-and-influenza-co-morbidity-in-c57bl6j-mice

Research

JoVE Journal

Biology

1.5K Views.  University of Tennessee Health Science Center. Asthma and influenza are diseases affecting the pulmonary system that affects millions worldwide. The aim of this study was to develop a mouse model of asthma and influenza comorbidity to study the intersection of these two diseases in the same host. 

Video: Modeling Asthma and Influenza Co-morbidity in C57BL/6J Mice

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Murine Colitis Modeling using Dextran Sulfate Sodium (DSS)

Colitis can occur from viral or bacterial infections, ischemic insult, or autoimmune disorders; most notably Ulcerative Colitis and the colonic variant of Crohn&rsquo;s Disease - Crohn&rsquo;s Colitis. Acute colitis may present with abdominal pain and distention, malabsorption, diarrhea, hematochezia and mucus in the stool. We are beginning to understand the complex interactions between the environment, genetics, and epithelial barrier dysfunction in Inflammatory Bowel Disease and animal models of colitis have been essential in advancing our understanding of this disease. One popular model involves supplementing the drinking water of mice with low-molecular weight Dextran Sodium Sulfate (DSS), resulting in epithelial damage and a robust inflammatory response in the colon lasting several days 1.Variations of this approach can be used to model acute injury, acute injury followed by repair, and repeated cycles of DSS interspersed with recovery modeling chronic inflammatory diseases 2. After a single four-day treatment of 3% DSS in drinking water, mice show signs of acute colitis including weight loss, bloody stools, and diarrhea. Mice are euthanized at the conclusion of the treatment course and at necropsy dissected colons are processed and can be 'Swiss rolled" 3 to allow microscopic analysis of the entire colon or infused with formalin as "sausages" to allow macroscopic analysis. Tissue is then embedded in paraffin, sectioned, and stained for histologic review.

Murine Colitis Modeling using Dextran Sulfate Sodium DSS

Dextran sulfate sodium (DSS) administered in the drinking water is an established murine inflammatory injury model of acute colitis. This protocol outlines the method for DSS treatment and the preparation of tissues.

Colitis can occur from viral or bacterial infections, ischemic insult, or autoimmune disorders; most notably Ulcerative Colitis and the colonic variant of ...

Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart failure.1 TAC initially leads to compensated hypertrophy of the heart, which often is associated with a temporary enhancement of cardiac contractility. Over time, however, the response to the chronic hemodynamic overload becomes maladaptive, resulting in cardiac dilatation and heart failure.2 The murine TAC model was first validated by Rockman et al.1, and has since been extensively used as a valuable tool to mimic human cardiovascular diseases and elucidate fundamental signaling processes involved in the cardiac hypertrophic response and heart failure development. When compared to other experimental models of heart failure, such as complete occlusion of the left anterior descending (LAD) coronary artery, TAC provides a more reproducible model of cardiac hypertrophy and a more gradual time course in the development of heart failure. Here, we describe a step-by-step procedure to perform surgical TAC in mice. To determine the level of pressure overload produced by the aortic ligation, a high frequency Doppler probe is used to measure the ratio between blood flow velocities in the right and left carotid arteries.3, 4 With surgical survival rates of 80-90%, transverse aortic banding is an effective technique of inducing left ventricular hypertrophy and heart failure in mice.

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model to study mechanisms underlying cardiac hypertrophy and heart failure development. Here, we describe procedures to constrict the aorta to create a reproducible degree of cardiac hypertrophy in mice.

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart ...

In situ Quantification of Pancreatic Beta-cell Mass in Mice

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. The process of monitoring pancreatic beta-cell proliferation and islet growth is particularly challenging. We have developed a method to capture the distribution of beta-cells in the intact pancreas of transgenic mice with fluorescence-tagged beta-cells with a macro written for ImageJ (rsb.info.nih.gov/ij/). Following pancreatic dissection and tissue clearing, the entire pancreas is captured as a virtual slice, after which the GFP-tagged beta-cells are examined. The analysis includes the quantification of total beta-cell area, islet number and size distribution with reference to specific parameters and locations for each islet and for small clusters of beta-cells. The entire distribution of islets can be plotted in three dimensions, and the information from the distribution on the size and shape of each islet allows a quantitative and qualitative comparison of changes in overall beta-cell area at a glance.

In situ Quantification of Pancreatic Beta-cell Mass in Mice

The following protocol outlines the process of pancreatic dissection for virtual slice imaging, and the subsequent quantification of all GFP-tagged beta-cells in the entire pancreas.

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. The process of monitoring pancreatic ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Evaluation of Respiratory System Mechanics in Mice using the Forced Oscillation Technique

The forced oscillation technique (FOT) is a powerful, integrative and translational tool permitting the experimental assessment of lung function in mice in a comprehensive, detailed, precise and reproducible manner. It provides measurements of respiratory system mechanics through the analysis of pressure and volume signals acquired in reaction to predefined, small amplitude, oscillatory airflow waveforms, which are typically applied at the subject's airway opening. The present protocol details the steps required to adequately execute forced oscillation measurements in mice using a computer-controlled piston ventilator (flexiVent; SCIREQ Inc, Montreal, Qc, Canada). The description is divided into four parts: preparatory steps, mechanical ventilation, lung function measurements, and data analysis. It also includes details of how to assess airway responsiveness to inhaled methacholine in anesthetized mice, a common application of this technique which also extends to other outcomes and various lung pathologies. Measurements obtained in na&iuml;ve mice as well as from an oxidative-stress driven model of airway damage are presented to illustrate how this tool can contribute to a better characterization and understanding of studied physiological changes or disease models as well as to applications in new research areas.

The present protocol provides a detailed step-by-step description of the procedures required to execute measurements of respiratory system mechanics as well as the assessment of airway responsiveness to inhaled methacholine in mice using the forced oscillation technique (flexiVent; SCIREQ Inc, Montreal, Qc, Canada).

The  forced oscillation technique (FOT) is a powerful, integrative and translational  tool permitting the experimental assessment of lung function in mice ...

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) 1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.
The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency 3. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction 4. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues 5. This later change increases muscle stiffness 6. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Changes in limb muscle contractile and passive mechanical properties are important biomarkers for muscle diseases. This manuscript describes physiological assays to measure these properties in the murine extensor digitorum longus and tibialis anterior muscles.

Body  movements are mainly provided by mechanical function of skeletal muscle. Skeletal  muscle is composed of numerous bundles of myofibers that are ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter implantation. The technical limitation of this surgical technique lies in the finesse of the hands. The injection is rapid, especially for a trained experimenter, and since tissue disruption with this technique is minimal, repeated injections are possible; moreover immune reaction to foreign tools (e.g. catheter) does not occur, thereby giving a better and more specific read out of spinal cord modulation. Since the application of the substance is largely limited to the target region of the spinal cord, drugs do not need to be applied in large dosages, and more importantly unwanted effects on other tissue, as observed with a systemic delivery, could be circumvented1,2. Moreover, we combine this technique with in vivo transfection of nucleic acid with the help of polyethylenimine (PEI) reagent3, which provides tremendous versatility for studying spinal functions via delivery of pharmacological agents as well as gene, RNA, and protein modulators.

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a simple and rapid technique of intrathecal needle puncture for a localized transfection of siRNA in the lumbar spinal cord in mouse under short lasting light anesthesia.

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...