This work&#160;was funded by NIH P20GM113123 and UND SMHS pilot grant.

The protocol should be performed in a sterile condition under a biosafety level 2 laminar flow hood (biosafety cabinet). All the medium and solutions should be thawed at 37 &#176;C prior to use. Centrifugation should be performed at 4 &#176;C. All the materials used in the experiment must be sterile. The primary cells (deidentified donor) were obtained in collaboration with Dr. Kristina Bailey at the University of Nebraska Medical Center (UNMC), Omaha, NE. The primary cells are from deidentified donors, and the cells do not fall under the NIH Human Subject. The cells were obtained under an approved Material Transfer Agreement (MTA) between the University of North Dakota, Grand Forks, ND, and UNMC, Omaha, NE. Here, we used passaged 4 NHBE cells. Although higher passage NHBE cells (up to passage 8) were also shown to differentiate into airway epithelium, we routinely use passage four NHBE cells because there is no obvious difference in whole genome transcription profiles among passages 1 to 46,14.
1. Primary cell culture
NOTE: Careful handling of the primary cells is crucial. The whole protocol should avoid harsh treatment and excessive pipetting of the primary cells.
<ol>
	<li>Using a sterile serological pipette add 5 mL of collagen to a 100 mm tissue culture (TC) dish to pre-coat the dish. Incubate the TC dish with collagen at room temperature (RT) for 20 min.</li>
	<li>Using a sterile serological pipette, aspirate the collagen from the TC dish and replace it with 8 mL of pre-thawed complete airway epithelial cell (AEC) medium supplemented with 2% (v/v) penicillin-streptomycin and 1% (v/v) amphotericin B (Table 1).</li>
	<li>Thaw one vial (containing 5 x 105 cells) of primary normal human bronchial epithelial (NHBE) cells in a water bath at 37 &#176;C.</li>
	<li>With a 5 mL sterile serological pipette, add 1 mL of pre-thawed complete AEC medium to the cells dropwise to acclimatize the NHBE cells, and collect all the cells in a sterile 15 mL conical tube.</li>
	<li>Pellet the cells by centrifugation at 212 x g for 5 min at 4 &#176;C. Gently discard the medium from the tube and resuspend the cells in the leftover medium drops in the tube by gentle tapping. 
	NOTE: Excessive and harsh tapping should be avoided.</li>
	<li>Using a 5 mL sterile serological pipette, slowly add 2 mL of complete AEC medium to the resuspended cells and collect all the cells.</li>
	<li>Add the cells to the plate pre-added with 8 mL of complete AEC medium. Move the TC dish sideways to distribute the cells in the dish uniformly.</li>
	<li>Incubate the cells at 37 &#176;C in an 85%-95% humid CO2 cell culture incubator. After 24 h, observe the NHBE cells under the microscope at 10x or 20x for their attachment to the TC dish and growth. If the cells are suspended in the medium, this shows dead cells.</li>
	<li>After 48 h, aspirate the old medium from the TC dish with a sterile serological pipette.</li>
	<li>Using a sterile serological pipette, add 10 mL of pre-warmed, complete AEC medium to the cells. Add the media slowly to one corner; avoid direct pouring over the top of the cells.</li>
	<li>Change the complete AEC media every 48 h until the cells reach 95%-100% confluency.</li>
</ol>
2. Air-Liquid interface culture
<ol>
	<li>Using a P200 pipette, add 100 &#181;L of collagen to a pre-coated 6.5 mm transwell insert with 0.4 &#956;m-pore polyester&#160;membrane (from here on referred as inserts) and incubate at RT for 20 min.</li>
	<li>With a sterile serological pipette, aspirate the medium from the confluent NHBE cells.</li>
	<li>Add 5 mL of 1x Dulbecco&#39;s Phosphate Buffer Saline (DPBS) with a sterile serological pipette to one corner of the TC dish.</li>
	<li>Gently swirl the plate to wash the cells and aspirate the 1x DPBS with a sterile serological pipette. Ensure cells do not dry out at any step during the protocol.</li>
	<li>Using a serological pipette, add 5 mL of TrypsinLE to the cells and incubate at 37 &#176;C in a humid CO2 cell culture incubator until all the cells are dissociated from the bottom surface of the plate.</li>
	<li>Using a sterile serological pipette, collect all the cells in a 15 mL conical tube. With a sterile serological pipette, add 3 mL of trypsin to the TC dish and collect any residual cells.</li>
	<li>Pellet the cells by centrifugation at 212 x g for 5 min at 4 &#176;C. Discard the medium and gently tap the tube to resuspend the cells in the leftover trypsin drops in the tube.</li>
	<li>Using a serological pipette add 2 mL of complete AEC medium to the resuspended cells.</li>
	<li>Count the cells using a hemacytometer or automated cell counter.</li>
	<li>Dilute the cells with complete AEC media, with each insert receiving 5 x 104 cells in a volume of 200 &#181;L.</li>
	<li>With sterile forceps, transfer the inserts to an empty well in a 24-well culture dish.</li>
	<li>Using a P1000 pipette, add 500 &#181;L of complete AEC media to the basal well of the plate.</li>
	<li>Using sterile forceps, transfer the inserts back to the basal well with added complete AEC media.</li>
	<li>With a P200 pipette, add 5 x 104 resuspended NHBE cells in 200 &#181;L of the complete AEC medium to the apical side of the insert. Incubate the cells at 37 &#176;C in an 85% - 95% humid CO2 cell culture incubator.</li>
	<li>After 24 h, observe the cells under the microscope to see if they have formed a confluent layer in the well. 
	NOTE: If the cells do not form a confluent layer, change the basal AEC medium every 48 h with fresh, complete AEC medium till the cells form a confluent monolayer. While changing basal media, transfer the inserts to the empty well with sterile forceps. Aspirate the old medium with a P1000 pipette, add fresh medium, and place the inserts back in fresh, complete AEC medium.</li>
	<li>Once the cells are confluent, transfer the inserts to an empty well with sterile forceps. Using a P1000 pipette, aspirate all the basal AEC medium.</li>
	<li>With a P1000 pipette, add 500 &#181;L of fresh complete air-liquid interface differentiation (ALI) medium supplemented with 2% (v/v) penicillin-streptomycin and 1% (v/v) amphotericin B to the basal well.&#160;</li>
	<li>Transfer the inserts to the basal well with added complete ALI medium using sterile forceps. Using a P200 pipette, gently aspirate the apical AEC medium from the well. 
	NOTE: The apical side should not be added with the complete ALI medium. Aspirate the AEC medium carefully from the apical side to avoid any cell detachment.</li>
	<li>Incubate the cells at 37 &#176;C in a humid CO2 cell culture incubator. After 48 h, transfer the inserts to an empty well with sterile forceps.</li>
	<li>Using a P1000 pipette, aspirate the old medium and add fresh 500 &#181;L of complete ALI medium.</li>
	<li>With sterile forceps, place the inserts back in the well with fresh medium. Keep changing the complete ALI medium every 48 h till day 28 (four weeks).</li>
	<li>On day 28, using a P200 pipette, add 100 &#181;L of 1x DPBS to the apical side of the inserts and incubate at 37 &#176;C in a humid CO2 cell culture incubator for 30 min. Aspirate the 1x DPBS with a P200 pipette. 
	NOTE: DPBS should be added by&#160;pouring&#160;on the wall&#160;instead of directly to the cells. While aspirating DPBS, avoid touching the epithelium and vigorous pipetting because any damage to the epithelium can compromise the epithelium (tissue-like) integrity.</li>
	<li>From the 2nd week of differentiation, evaluate mucus production by observing a possible darker layer due to mucus on the epithelium under the microscope. The excessive mucus can be removed by DPBS washing. Briefly, add 200 &#181;L of 1x DPBS on the apical side of the inserts on a 24-well plate and incubate the plate at 37 &#176;C in a humid CO2 cell culture incubator for 30 min. Remove DPBS and keep the plate at 37 &#176;C in a humid CO2 cell culture incubator.</li>
</ol>
3. Determination of ciliary beat frequency
NOTE: For determination of CBF at 37 &#176;C (if necessary), the microscope needs to have an environment chamber incubator providing 5% CO2 at 37 &#176;C and humidity.
<ol>
	<li>Turn on the microscope with its attached computer and the environment chamber. Open the door of the chamber to place the plate containing the inserts on the microscope stage.</li>
	<li>Once the plate is placed on the stage, close the chamber. Using the microscope stage operating knob, move the plate to bring the inserts into view.</li>
	<li>Select the 20x magnification lens in the phase-contrast mode. Observe the plate through the eyepiece of the microscope and focus the cell using the tuning knob.</li>
	<li>On the computer screen, click on SAVA system icon. SAVA program should be installed on the computer connected to the microscope.</li>
	<li>On the software interface, click on Configure Experiment. On the next tab, click on Create Experiment.</li>
	<li>A new tab will appear, give details of the directory where the data will be stored in the Enter Experiment Directory Name and experiment details in Enter Experiment Description, then click OK.</li>
	<li>Click The Experiment that was created and is shown in the Directory under the Experiments.</li>
	<li>Click on Modify and provide sample details in the Sample Description. Click on Add then on Exit to exit from this tab.</li>
	<li>On the main software interface on the experiment slot, select the experiment created from the dropdown menu on the main interface.</li>
	<li>Click on Record Video and monitor the ciliary beat frequency (CBF) on the computer screen. The screen will show focused ciliary movement.</li>
	<li>For each insert, record 6 different random fields for 2.1 s at 120 frames per second. Click on Save Video to monitor the CBF data.</li>
	<li>To analyze data, click on Analyse Video on the software interface. On the next tab, click on OK.</li>
	<li>On the following tab, click on Analyse All. Download the spreadsheet generated for every experiment in the computer SAVA data acquisition folder in the computer.</li>
	<li>Take Gaussian mean frequency for data presentation.</li>
</ol>
4. Quantification of transepithelial electrical resistance
NOTE: TEER quantification needs liquid at the apical side of the airway epithelium. To establish this protocol, add 100 &#181;L of 1x DPBS at the apical side of the airway epithelium in the inserts that already contain the basal medium (experimental) or 500 &#181;L of 1x DPBS.
<ol>
	<li>Turn on the EVOM2 volt ohmmeter using the power switch. Connect the Test Resistor to the EVOM2 at the INPUT slot.</li>
	<li>Monitor the reading on the EVOM2 screen. If it is above or below 1000, then it needs calibration. Using forceps, turn the ADJ switch at the EVOM2 volt ohmmeter till it shows a uniform 1000 reading.</li>
	<li>Take an empty insert for experimental control reading. Using a P200 pipette add 100 &#181;L of 1x DPBS at the apical side of the empty insert, while 500 &#181;L of 1x DPBS is added at the basal side with a P1000 pipette.</li>
	<li>Disconnect the Test Resistor and connect the STX2 electrode at the INPUT slot on EVOM2. Gently clean the STX2 electrode with 70% ethanol.</li>
	<li>Insert the STX2 electrode in the emptyinsert with 1x DPBS, keeping the shorter electrode leg on the apical part of each insert and the longer electrode leg on the basal part.</li>
	<li>Record the stable reading on the EVOM2 screen, as the reading can fluctuate due to movement or other undetermined reasons.</li>
	<li>Insert the STX2 electrode in the insert with 28-day differentiated epithelium, keeping the shorter electrode leg on the apical part of each insert and the longer electrode leg on the basal part in the medium.</li>
	<li>Record the stable reading on the EVOM2 screen. Take several independent readings to calculate a reliable average. Subtract the background (empty insert reading) from the experimental insert reading for calculation.</li>
</ol>

Assessing Biophysical Properties of Differentiated Airway Epithelium 

<ol>
	<li>GBDCRD Collaborators. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+burden+of+chronic+respiratory+diseases+and+risk+factors,+1990-2019:+an+update+from+the+Global+Burden+of+Disease+Study+2019.">Global burden of chronic respiratory diseases and risk factors, 1990-2019: an update from the Global Burden of Disease Study 2019.</a> E Clin Med. 59, 101936 (2023).</li><li>Fendrick, A. M., Monto, A. S., Nightengale, B., Sarnes, 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+economic+burden+of+non-influenza-related+viral+respiratory+tract+infection+in+the+United+States.">The economic burden of non-influenza-related viral respiratory tract infection in the United States.</a> Arch Intern Med. 163 (4), 487-494 (2003).</li><li>LeMessurier, K. S., Tiwary, M., Morin, N. P., Samarasinghe, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+barrier+as+a+safeguard+and+regulator+of+defense+against+Influenza+A+virus+and+Streptococcus+pneumoniae.">Respiratory barrier as a safeguard and regulator of defense against Influenza A virus and Streptococcus pneumoniae.</a> Front Immunol. 11, 3 (2020).</li><li>Plasschaert, L. W., 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=A+single-cell+atlas+of+the+airway+epithelium+reveals+the+CFTR-rich+pulmonary+ionocyte.">A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte.</a> Nature. 560 (7718), 377-381 (2018).</li><li>Calven, J., Ax, E., Radinger, 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+airway+epithelium-A+central+player+in+Asthma+pathogenesis.">The airway epithelium-A central player in Asthma pathogenesis.</a> Int J Mol Sci. 21 (23), 8907 (2020).</li><li>Talukdar, S. N., 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=RSV-induced+expanded+ciliated+cells+contribute+to+bronchial+wall+thickening.">RSV-induced expanded ciliated cells contribute to bronchial wall thickening.</a> Virus Res. 327, 199060 (2023).</li><li>Osan, 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=Goblet+cell+hyperplasia+increases+SARS-CoV-2+infection+in+chronic+obstructive+pulmonary+disease.">Goblet cell hyperplasia increases SARS-CoV-2 infection in chronic obstructive pulmonary disease.</a> Microbiol Spectr. 10 (4), e0045922 (2022).</li><li>Srinivasan, B., 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=TEER+measurement+techniques+for+in+vitro+barrier+model+systems.">TEER measurement techniques for in vitro barrier model systems.</a> J Lab Autom. 20 (2), 107-126 (2015).</li><li>Bustamante-Marin, X. M., Ostrowski, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cilia+and+mucociliary+clearance.">Cilia and mucociliary clearance.</a> Cold Spring Harb Perspect Biol. 9 (4), a028241 (2017).</li><li>Rhee, C. 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=Ciliary+beat+frequency+in+cultured+human+nasal+epithelial+cells.">Ciliary beat frequency in cultured human nasal epithelial cells.</a> Ann Otol Rhinol Laryngol. 110 (11), 1011-1016 (2001).</li><li>Scopulovic, L., Francis, D., Pandzic, E., Francis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+cilia+beat+frequency+using+high-speed+video+microscopy:+Assessing+frame+rate+requirements+when+imaging+different+ciliated+tissues.">Quantifying cilia beat frequency using high-speed video microscopy: Assessing frame rate requirements when imaging different ciliated tissues.</a> Physiol Rep. 10 (11), e15349 (2022).</li><li>Jing, J. C., Chen, J. J., Chou, L., Wong, B. J. F., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+and+detection+of+ciliary+beating+pattern+and+frequency+in+the+upper+airway+using+phase+resolved+Doppler+optical+coherence+tomography.">Visualization and detection of ciliary beating pattern and frequency in the upper airway using phase resolved Doppler optical coherence tomography.</a> Sci Rep. 7 (1), 8522 (2017).</li><li>Yuksel, H., Turkeli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airway+epithelial+barrier+dysfunction+in+the+pathogenesis+and+prognosis+of+respiratory+tract+diseases+in+childhood+and+adulthood.">Airway epithelial barrier dysfunction in the pathogenesis and prognosis of respiratory tract diseases in childhood and adulthood.</a> Tissue Barriers. 5 (4), e1367458 (2017).</li><li>Rayner, R. E., Makena, P., Prasad, G. L., Cormet-Boyaka, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+Normal+Human+Bronchial+Epithelial+(NHBE)+Cell+3D+Cultures+for+in+vitro+Lung+Model+Studies.">Optimization of Normal Human Bronchial Epithelial (NHBE) Cell 3D Cultures for in vitro Lung Model Studies.</a> Sci Rep. 9 (1), 500 (2019).</li><li>Osan, J. K., DeMontigny, B. A., Mehedi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry+for+protein+detection+in+PFA-fixed+paraffin-embedded+SARS-CoV-2-infected+COPD+airway+epithelium.">Immunohistochemistry for protein detection in PFA-fixed paraffin-embedded SARS-CoV-2-infected COPD airway epithelium.</a> STAR Protoc. 2 (3), 100663 (2021).</li><li>Talukdar, S. N., McGregor, B., Osan, J. K., Hur, J., Mehedi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+Syncytial+Virus+Infection+Does+Not+Induce+Epithelial-Mesenchymal+Transition.">Respiratory Syncytial Virus Infection Does Not Induce Epithelial-Mesenchymal Transition.</a> J Virol. 97 (7), e0039423 (2023).</li><li>Christopher, A. B., 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+effects+of+temperature+and+anesthetic+agents+on+ciliary+function+in+murine+respiratory+epithelia.">The effects of temperature and anesthetic agents on ciliary function in murine respiratory epithelia.</a> Front Pediatr. 2, 111 (2014).</li><li>Smith, C. 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Authors have no conflict of interest.

Human primary airway epithelium (ALI culture) is increasingly used as an in vitro lung model for biological assessments to investigate function and mechanisms and to reduce animal use14. ALI has several advantages over any monolayer submerged culture. For example, it provides a tissue-like pseudostratified airway epithelium that mimics the apical side of the lung airway6,7,14. However, the quality and int...

Chronic respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis are some of the major health concerns worldwide1. The prevalence of respiratory diseases caused by viruses such as human influenza A virus, respiratory syncytial virus (RSV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also cause economic and public health burden2. Therefore, there is an immense need to develop treatment regimens for respiratory illnesses that lead to irreversible respiratory tissue damage. The respiratory epithelial tissue itself is not only involved in oxygen uptake but also provides a barrier to protect the body from invading pathogens and hazardous chemicals3. The respiratory epithelium has a complex cellular organization composed of three major cell types: ciliated cells, goblet cells, and basal cells. Recently, there has been a report on the presence of novel but rare ionocyte cells in the airway epithelium4. The complex tissue functions in a coordinated fashion to provide innate immune responses such as secretion of antimicrobial peptides, cytokines release to activate an adaptive immune response, and mucociliary clearance5. The lack of a suitable airway model is one of the obstacles to the study of respiratory infections and the development of treatments.
The air-liquid interface (ALI) model is becoming a key tool for research on respiratory diseases6. It is an effective in vitro lung airway model as the primary lung airway cells differentiate into a pseudostratified airway epithelium composed of at least three types of cells that generally reside at the apical side of the airway. First, ciliated cells cover the majority of the apical side of the airway and contribute to mucociliary clearance. Second, goblet cells produce mucus and are the largest cells that co-reside with ciliary cells at the apical side of the airway. Third, basal cells reside at the basal layer of the airway and are the progenitor cells that differentiate into different epithelial cells5,7. Although ionocytes and tuft cells are rare in the airway epithelium, they may also play a role in ciliary function and membrane permeability4. This technique differs from the traditional submerged cell culture, which does not mimic the in vivo lung environment. Since ALI is produced from primary epithelial cells derived from healthy people, patients with asthma, and COPD, it offers a more diverse platform for studying responses to infection and disease pathophysiology.
The health assessment of the ALI-developed cultures is an essential aspect to monitor, ensuring the cultured cells&#39; viability, functionality, and physiological relevance. It enhances the confidence in the integrity, reliability, and reproducibility of ALI cultures. As previously published, our group has been evaluating the ALI culture integrity by assessing two biophysical parameters: ciliary function by quantifying ciliary beat frequency (CBF) and epithelial barrier integrity by determining transepithelial electrical resistance (TEER)6,7,8. Mucociliary clearance (MCC) is one of the important features of the airway epithelium carried by ciliated cells. Specialized organelles called cilia on the surface of ciliated cells beat in metachronal waves to clear the airways of infections and inhaled particles stuck in the mucous layer of the airway epithelium. Effective MCC is totally dependent upon proper ciliary activity9. A good CBF is indicative of a healthy, intact airway epithelium; hence, monitoring of the CBF for ALI cultures provides valuable insight into the epithelium&#39;s integrity10. Although there are multiple ways to quantify CBF, for example, high-speed video microscopy11 and phase-resolved Doppler optical coherence tomography12, each method needs technical expertise and specialized equipment. In this protocol, we provide an easier and less -technical way of quantifying CBF of the differentiated airway epithelium. We also described a method for quantifying TEER of the same epithelium, which indicates epithelial tissues&#39; integrity and barrier function13.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1x DPBS</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Airway Epithelial Cell Growth Medium</td>
			<td>PromoCell, Germany</td>
			<td>C-21060</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>15290-026</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOM2 volt Ohmmeter</td>
			<td>World Precision Instruments, USA</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Solution</td>
			<td>STEMCELL technologies, USA</td>
			<td>07980</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone stock</td>
			<td>STEMCELL technologies, USA</td>
			<td>07926</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope: Leica DMI8 (Leica Microscope) with 120f/sec high-speed camera (Basler) associated with the microscope. Microsope also fixed with stage chamber incubator i8.</td>
			<td>Leica, USA</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Corning, USA</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>PneumaCult-ALI Basal Medium&#160;</td>
			<td>STEMCELL technologies, USA</td>
			<td>05002</td>
			<td></td>
		</tr>
		<tr>
			<td>PureCol</td>
			<td>Advanced BioMatrix, USA</td>
			<td>5005</td>
			<td></td>
		</tr>
		<tr>
			<td>Sisson-Ammons video analysis (SAVA) system. Software V.2.1.15&#160;</td>
			<td>Amson Engineering, USA</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
	</tbody>
</table>

determining ciliary function and membrane impermeability of the pseudostratified lung airway epithelium

Differentiating primary lung airway epithelial cells in the air-liquid interface (ALI) is a popular technique to develop a multi-cellular pseudostratified airway epithelium that mimics the apical side of the lung airway. While the differentiation of primary lung airway cells is expected, the assessment of biophysical properties like ciliary function and membrane impermeability provides a quality assessment of the airway epithelium and ensures the reliability of the experiment. Here, we describe a straightforward protocol for the development of multi-cellular pseudostratified airway epithelium in ALI culture and assess two important biophysical properties: ciliary function and membrane impermeability. To determine ciliary function, we captured the ciliary movement of a 4-week differentiated airway epithelium using a high-speed camera attached to an inverted microscope, followed by quantifying ciliary beat frequency (CBF) using the Simon Amon Video Analysis (SAVA) system. We also measured transepithelial electrical resistance (TEER) using a volt-ohm meter to determine the epithelial barrier integrity of the airway epithelium.

We evaluated both ciliary function and membrane impermeability of the airway epithelium obtained from differentiating primary NHBE cells from two independent deidentified donors (two nonsmoking, healthy adults or adults with COPD, above 50-year-old females). For determining ciliary function, we determined the CBF of the 28-day differentiated airway epithelium. We observed CBF reached at least 3 Hz for both airway epithelium, which was comparable to the previously published results (Figure 1)...

Determining Ciliary Function and Membrane Impermeability of the Pseudostratified Lung Airway Epithelium

Air-liquid interface culture is commonly used to develop pseudostratified airway epithelium by differentiating primary normal human bronchial epithelial cells that mimic the apical side of the lung airway. Here, we describe an easy protocol for determining its quality by monitoring its biophysical properties, such as ciliary function and membrane integrity.

Differentiating primary lung airway epithelial cells in the air-liquid interface (ALI) is a popular technique to develop a multi-cellular pseudostratified ...

determining-ciliary-function-membrane-impermeability-pseudostratified

Research

JoVE Journal

Biology

379 Views.  University of North Dakota. Air-liquid interface culture is commonly used to develop pseudostratified airway epithelium by differentiating primary normal human bronchial epithelial cells that mimic the apical side of the lung airway. Here, we describe an easy protocol for determining its quality by monitoring its biophysical properties, such as ciliary function and membrane integrity.

Video: Determining Ciliary Function and Membrane Impermeability of the Pseudostratified Lung Airway Epithelium

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

Determining the Contribution of the Energy Systems During Exercise

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given physical activity. Although some sports are relatively easy to be reproduced in a laboratory (e.g., running and cycling), a number of sports are much more difficult to be reproduced and studied in controlled situations. This method presents how to assess the differential contribution of the energy systems in sports that are difficult to mimic in controlled laboratory conditions. The concepts shown here can be adapted to virtually any sport.
The following physiologic variables will be needed: rest oxygen consumption, exercise oxygen consumption, post-exercise oxygen consumption, rest plasma lactate concentration and post-exercise plasma peak lactate. To calculate the contribution of the aerobic metabolism, you will need the oxygen consumption at rest and during the exercise. By using the trapezoidal method, calculate the area under the curve of oxygen consumption during exercise, subtracting the area corresponding to the rest oxygen consumption. To calculate the contribution of the alactic anaerobic metabolism, the post-exercise oxygen consumption curve has to be adjusted to a mono or a bi-exponential model (chosen by the one that best fits). Then, use the terms of the fitted equation to calculate anaerobic alactic metabolism, as follows: ATP-CP metabolism = A1 (mL . s-1) x t1 (s). Finally, to calculate the contribution of the lactic anaerobic system, multiply peak plasma lactate by 3 and by the athlete&#x2019;s body mass (the result in mL is then converted to L and into kJ).
The method can be used for both continuous and intermittent exercise. This is a very interesting approach as it can be adapted to exercises and sports that are difficult to be mimicked in controlled environments. Also, this is the only available method capable of distinguishing the contribution of three different energy systems. Thus, the method allows the study of sports with great similarity to real situations, providing desirable ecological validity to the study.

This protocol allows researchers focused on exercise and sports sciences to determine the relative contribution of three different energy systems to the total energy expenditure during a large variety of exercises.

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated to generate a carbon/platinum &#8220;cast&#8221; intended for examination by transmission electron microscopy. Specimens are subjected to ultrarapid freezing rates, often in the presence of cryoprotective agents to limit ice crystal formation, with subsequent fracturing of the specimen at liquid nitrogen cooled temperatures under high vacuum. The resultant fractured surface is replicated and stabilized by evaporation of carbon and platinum from an angle that confers surface three-dimensional detail to the cast. This technique has proved particularly enlightening for the investigation of cell membranes and their specializations and has contributed considerably to the understanding of cellular form to related cell function. In this report, we survey the instrument requirements and technical protocol for performing freeze-fracture, the associated nomenclature and characteristics of fracture planes, variations on the conventional procedure, and criteria for interpretation of freeze-fracture images. This technique has been widely used for ultrastructural investigation in many areas of cell biology and holds promise as an emerging imaging technique for molecular, nanotechnology, and materials science studies.

Basic techniques and refinements of freeze-fracture processing of biological specimens and nanomaterials for examination by transmission electron microscopy are described. This technique is a preferred method for revealing ultrastructural features and specializations of biological membranes and for obtaining ultrastructural level dimensional and spatial data in materials sciences and nanotechnology products.

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated ...

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods are needed that can quantify the pathologic changes. Furthermore, to provide translational relevance to the mouse models, such measurements should be tests that can easily be done in both humans and mice. Unfortunately, in the present literature few phenotypic measurements of lung function have direct application to humans. One exception is the diffusing capacity for carbon monoxide, which is a measurement that is routinely done in humans. In the present report, we describe a means to quickly and simply measure this diffusing capacity in mice. The procedure involves brief lung inflation with tracer gases in an anesthetized mouse, followed by a 1 min gas analysis time. We have tested the ability of this method to detect several lung pathologies, including emphysema, fibrosis, acute lung injury, and influenza and fungal lung infections, as well as monitoring lung maturation in young pups. Results show significant decreases in all the lung pathologies, as well as an increase in the diffusing capacity with lung maturation. This measurement of lung diffusing capacity thus provides a pulmonary function test that has broad application with its ability to detect phenotypic structural changes with most of the existing pathologic lung models.

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

We describe a means to quickly and simply measure the lung diffusing capacity in mice and show that it is sufficiently sensitive to phenotype changes in multiple common lung pathologies. This metric thus brings direct translational relevance to the mouse models, since diffusing capacity is also easily measured in humans.

The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods ...

Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and exercise. However, these approaches do not address the underlying causes of balance decline. Using mice, the impact of near infrared light (NIr) on the metabolism of cells in the vestibular sensory epithelium was assessed. Data collected shows that this simple and safe intervention may protect these vulnerable cells from the deleterious effects of natural aging. mRNA was extracted from the isolated peripheral vestibular sensory epithelium (crista ampullaris and utricular macula) and subsequently transcribed into a cDNA library. This library was then probed for the expression of ubiquitous antioxidant (SOD-1). Antioxidant gene expression was then used to quantify cellular metabolism. Using transcranial delivery of NIr in young (4 weeks) and older (8 - 9 months) mice, and a brief treatment regime (90 sec/day for 5 days), this work suggests NIr alone may be sufficient to improve mitochondrial function in the vestibular sensory epithelium. Since there are currently no available, affordable, non-invasive methods of therapy to improve vestibular hair cell function, the application of external NIr radiation provides a potential strategy to counteract the impact of aging on cellular metabolism inthe vestibular sensory epithelium.

Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Mitochondrial dysfunction is a hallmark of cellular senescence. This paper uses non-invasive near-infrared (NIr) treatment to improve mitochondrial function in the aging mouse vestibular sensory epithelium.

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and ...

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.

Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP.

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established ...

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-P&#233;rot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

Presented here is a protocol to assess the contractile properties of striated muscle myofibrils with nano-Newton resolution. The protocol employs a setup with an interferometry-based, optical force probe. This setup generates data with a high signal-to-noise ratio and enables the assessment of the contractile kinetics of myofibrils.