Measurement of Nitric Oxide Production in Mouse Tracheal Epithelial Cell Cultures Differentiated at the Air Liquid Interface

Summary

This protocol describes a method to quantify nitric oxide (NO) production in airway epithelial cultures differentiated at the air–liquid interface (ALI) by measuring nitrite in apical secretions using a triiodide-based chemiluminescence assay.

Abstract

Airway epithelial cultures differentiated at the air–liquid interface (ALI) provide a physiologically relevant model to study nitric oxide (NO) signaling in ciliated cells. Here, we describe a protocol to quantify NO production by measuring nitrite (NO₂⁻), a stable oxidation product of NO, in samples collected from ALI-differentiated mouse tracheal epithelial cells. Apical washes, basal media and cell lysates are collected and analyzed using a triiodide-based chemiluminescence assay coupled to a nitric oxide analyzer (NOA). Upon injection into the reaction vessel, nitrite is chemically reduced to NO, which is detected by ozone-based chemiluminescence. Signal intensity is quantified and converted to picomoles of nitrite using a standard calibration curve, and values are normalized to total cellular protein content. Assay performance is validated using pharmacological modulation of NO levels. Upon treatment with the NO donor Diethylenetriamine NONOate (DETA-NONOate) and the NO synthase inhibitor N(ω)-Nitro-L-arginine methyl ester (L-NAME) we observe significant differences in nitrite levels across conditions. The most robust and reproducible signal is observed in apical washes, corresponding to the amount of NO released from the airway epithelium. This method reliably detects NO in samples from ALI-differentiated airway epithelia, providing an accurate in vitro platform to quantify NO production and model diseases associated with abnormal NO metabolism.

Introduction

A hallmark of several airway diseases, including asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF) and primary ciliary dyskinesia (PCD), is altered nitric oxide (NO) homeostasis measurable as changes in exhaled NO levels, which can largely vary in these diseases, being elevated in COPD and asthma^1,2, while drastically reduced in CF and PCD^3,4. These diseases commonly exhibit abnormalities in airway epithelial motile cilia, the key effectors of mucociliary clearance⁵. Because NO and its downstream signaling pathways are critical regulators of ciliary motility, understanding how NO production and signaling are altered in these contexts is essential for elucidating mechanisms of mucociliary dysfunction. Accordingly, a method that enables precise detection of NO production and release in airway epithelia provides a valuable tool for dissecting the regulation and physiological role of this signaling molecule. Such approaches are particularly useful in experimental settings where direct measurement of NO is challenging due to its short half-life and rapid reactivity.

NO regulates airway cilia biogenesis and function by influencing ciliated cell polarity and the coordinated beating of motile cilia^6,7,8. However, the mechanisms that maintain NO homeostasis in the airways remain incompletely understood. Airway epithelial cultures differentiated at air–liquid interface (ALI) provide a valuable in vitro system for investigating these mechanisms. At present, this model is widely used to study the impact of environmental insults on the airway epithelium^9,10,11 and to elucidate the pathophysiology of airway diseases¹². Compared with conventional submerged cultures^13,14, ALI–differentiated epithelia more faithfully reproduce the architecture and function of the native airway, including epithelial polarization, mucociliary differentiation, and realistic gas–liquid exchange, making them especially suitable to study apical NO release, signaling, and related ciliary function. This system is therefore well-suited for experiments aimed at quantifying NO-derived metabolites released into the airway lumen, particularly under conditions of epithelial differentiation or pharmacological manipulation.

Accurate measurement of NO concentration is essential, as both excessive and deficient NO levels can impair ciliary function. Currently, fluorescent probe–based methods (e.g., diaminofluoresceins, DAFs) available for detecting NO are only semi-quantitative and are limited by pH sensitivity, oxygen dependence and lack of specificity, as they can react with cellular oxidants and antioxidants, resulting in semi-quantitative measurements¹⁵. In addition, electrochemical sensors (e.g., NO-sensitive amperometric electrodes) enable real–time detection of NO¹⁶ but can be difficult to implement in complex biological systems due to NO short half–life, interference from redox-active species biofouling of the electrode surface, and challenges in maintaining stable calibration¹⁷. Alternative chemiluminescence-based approaches including direct gas-phase NO detection and reductive assays targeting different nitrogen oxide species, have also been described, but their application to airway epithelial cultures remains unexplored. These limitations highlight the need for sensitive, quantitative methods that can reliably detect low levels of NO-derived species in small-volume biological samples. To overcome these limitations, we established a protocol to quantify NO in apical secretions collected from ALI-differentiated airway epithelia using a Nitric Oxide Analyzer (NOA). The NOA is a highly sensitive chemiluminescence–based detector traditionally used to measure NO metabolites in chemical samples and more recently adapted for the analysis of biological specimens, including blood, tissue homogenates, and cell culture supernatants^7,18,19. NO is a highly reactive signaling molecule rapidly oxidized to nitrite in tissues under conditions of normal oxygen levels, with an exceptionally short half-life. Thus, nitrite as end product of NO oxidation serves as an indicator of total NO production in biological systems^20,21,22. This sensitive and reproducible chemiluminescence method allows detection of nitrite/NO at picomolar levels^23,24, making it ideal for measuring small changes in low–volume biological samples such as those derived from MTEC ALI cultures. However, this approach is best suited for conditions where nitrite accumulation reflects recent NO production and may be influenced by factors such as sample handling, background nitrite contamination, and epithelial differentiation state. Therefore, appropriate controls and careful experimental design are required to ensure accurate interpretation of results.

Protocol

All animal procedures were reviewed and approved by the University of Maryland Institutional Animal Care and Use Committee (Protocol # AUP–00004632).

NOTE: The protocol requires approximately 2 days for isolation of mice tracheal epithelial cells (MTEC), followed by 7–10 days for expansion, 5–6 days for proliferation in transwell inserts, 21 days for differentiation (time needed to acquire sufficient multi–ciliated cells), 24 h of drug treatment and one additional day for sample collection, NOA calibration, injections and data acquisition.

1. Preparation of media and solutions

NOTE: Refer to Table 1 for details on media ingredients.

1. Ham’s F–12 media
   1. Prepare the solution inside a sterile laminar flow hood. Add 5 mL of 100× Penicillin/Streptomycin antibiotics and 0.5 mL of Amphoterecin B (Fungizone) to 500 mL of Ham’s F12 medium in a sterile bottle.
   2. Mix by gently inverting the bottle 5–10 times to ensure complete mixing. Filter sterilize the solution using a 0.22 µm filter under sterile conditions, and store at 4 °C.
      ​CAUTION: Penicillin/streptomycin and amphotericin B are antimicrobial agents that may cause irritation or allergic reactions. Handle using appropriate personal protective equipment, including gloves and lab coat.
2. Pronase solution
   1. Add 15 mg of pronase to 10 mL of sterile Ham’s F12 medium in a sterile tube to obtain 0.15% pronase solution. Mix by gently inverting or pipetting up and down until the pronase is fully dissolved. Prepare fresh immediately before use.
3. DNase I solution
   1. Add 5 mg of crude pancreatic DNase I to 9 mL of Ham’s F12 medium and 1 mL of 10 mg/mL bovine serum albumin (BSA) solution in a sterile tube to obtain a 0.5 mg/mL DNase I solution.
   2. Gently mix by pipetting up and down until the DNase I is fully dissolved, avoiding bubble formation. Once fully dissolved, aliquot the solution into 1 mL sterile microcentrifuge tubes and store at -20 °C.
4. Ham’s F12 media with fetal bovine serum (FBS)
   1. Prepare the solution at room temperature inside a sterile laminar flow hood. Add 10 mL of fetal bovine serum (FBS) to 40 mL of Ham’s F12 media with antibiotics in a sterile conical tube to obtain 20% FBS in F12 medium.
   2. Mix gently by inverting the tube 5–10 times until the solution is homogeneous. Filter sterilize the solution using a 0.22 µm filter under sterile conditions, and store at 4 °C.
5. MTEC expansion media
   1. Prepare the MTEC expansion media at room temperature inside a sterile laminar flow hood by combining the components listed in Table 1 in a sterile bottle.
   2. Mix thoroughly by gently swirling or inverting the container until all components are fully combined. Filter sterilize the solution using a 0.22 µm filter under sterile conditions.
   3. Add ROCK inhibitor Y–27632 (5 mM stock solution) to a final concentration of 10 µM and Notch γ–secretase inhibitor DAPT (10 mM stock solution) to a final concentration of 5 µM immediately before use, and mix gently by inversion.
6. MTEC proliferation media
   1. Prepare the MTEC proliferation media at room temperature inside a sterile laminar flow hood by combining the components listed in Table 1.
   2. Mix thoroughly by gentle inversion or swirling until all components are fully combined. Filter sterilize the medium using a 0.22 µm filter under sterile conditions.
   3. Immediately before use, add ROCK inhibitor Y–27632 (5 mM stock solution) to a final concentration of 10 µM and retinoic acid (5 µM stock solution) to a final concentration of 0.05 µM. Mix gently by inversion before use.
7. MTEC differentiation media
   1. Prepare the MTEC differentiation medium at room temperature inside a sterile laminar flow hood by combining the components listed in Table 1 in a sterile container.
   2. Mix thoroughly by gentle inversion or swirling until all components are fully combined. Filter sterilize the medium using a 0.22 µm filter under sterile conditions.
   3. Immediately before use add retinoic acid to a final concentration of 0.05 µM and mix gently by inversion.

2. Isolation of mouse tracheal epithelial cells (MTEC)

1. Euthanize three adult mice (8–20 weeks old) by carbon dioxide (CO₂) inhalation in an approved euthanasia chamber according to institutional IACUC guidelines. Confirm death before tissue collection.
2. Place the animal dorsally (chest facing up) on an aseptic surface. Using clean forceps, gently lift the skin and cut it open with surgical scissors from the lower lip to the abdomen to expose the trachea.
3. Move the salivary glands aside and remove the connective tissue and trachealis muscle to expose the cartilage rings. Dissect the full length of trachea and place it in a Petri dish containing sterile Ham’s F12 medium. Combine three tracheas in one dish.
4. Transfer the dish into a laminar flow hood and place the tracheas to another dish with fresh Ham’s F12 medium. Remove any extratracheal tissue and cut the trachea longitudinally through the lumen.
5. Transfer all the tracheas into a 50 mL screw–cap conical tube containing sterile 10 mL 0.15% pronase solution. Incubate at 4 °C for 16–18 h.

3. Cell isolation and initial expansion

1. The next day invert the tube containing the tissues 10–15 times to dislodge epithelial cells and incubate at 4 °C for 45 min. Stop the pronase reaction by adding 10 mL of Ham’s media containing 20% FBS and mix by inverting 10–15 times.
2. Add 10 mL of Ham’s F12 medium with FBS to three 15 mL tubes. With a Pasteur pipette transfer the tracheal tissue sequentially from the 50 mL tube to the first, second and third 15 mL tube and invert 10–15 times in each tube to dislodge the tracheal epithelial cells. Discard the tissue after the final transfer.
3. Combine the whole media from the three 15 mL conical tubes with the medium in the 50 mL tube. Centrifuge at 360 × g for 10 min at 4 °C. Carefully remove the supernatant and add 0.5 mL of 0.5 mg/mL DNase I solution. Incubate on ice for 5 min.
4. Centrifuge at 360 × g for 5 min at 4 °C. Carefully discard the supernatant and resuspend the cell pellet in 7–10 mL of MTEC expansion medium.

4. Expansion and differentiation of MTEC

1. Prepare a collagen coated T75 cell culture flask using 10 mL of collagen I (rat tail) at 50 µg/mL and incubate at 37 °C for 16–18 h. Aspirate the collagen solution from the T75 flask and wash the flask three times with PBS (1×). Allow the flask to dry completely.
2. Seed the cell suspension consisting of 7–10 mL of MTEC expansion medium containing the cells into the collagen coated flask. Place the flask in an incubator at 37 °C and 5% CO₂. Replace the expansion medium every 2 or 3 days and monitor cell number until 70% confluency.
3. At 70% confluency, add 1 mL of Accutase solution and incubate for 10–15 min at 37 °C to detach the cell monolayer. Resuspend the cells in 10 mL of MTEC proliferation medium. Centrifuge at 1057 × g for 5 min at room temperature.
4. Discard the supernatant, resuspend the cells in proliferation medium and count the cells.
5. Seed 2 × 10⁵ cells in 0.5 mL volume per transwell insert (12 mm membrane diameter, 0.4 µm membrane pore size) precoated with collagen. Add 1.5 mL medium to the basal chamber of each insert and place the transwell in the incubator at 37 °C.
6. Change media in both the apical and basal chamber on day 3 and allow the cells to reach 100% confluency, usually by day 5 or 6. Include one insert without cells as a control.
7. Once confluent, wash the cells apically with 0.5 mL of MTEC differentiation media. Initiate air-liquid interface (ALI) by adding 0.75 mL of differentiation medium to the basal chamber only, leaving the cells in apical chamber exposed to air.
8. Every 2–3 days, wash cells in the apical surface with differentiation medium and replace basal medium. Allow cells to differentiate at ALI for 21 days.

5. Immunolabeling and imaging of ciliated epithelial cells

1. At ALI day 7, 14 and 21, fix the cells with 4% paraformaldehyde (PFA).
2. Perform immunolabelling of cells with anti–FoxJ1 antibody to detect FoxJ1, a transcription factor essential for motile ciliogenesis^25,26, and anti–acetylated α–tubulin antibody to detect acetylated α–tubulin, a structural marker of motile cilia according to published protocols^27,28,29.
   CAUTION: PFA is a hazardous chemical, so wear appropriate PPE (nitrile gloves and lab coat at a minimum) when fixing the cells.
3. Acquire images of the immunolabelled cells by confocal microscopy using a 40× or 60× objective. Collect Z–stacks spanning a depth of 10 µm with each step size of 0.5 µm.
4. Set laser power, detector gain and exposure settings to avoid signal saturation and maintain comparable acquisition parameters across samples.
5. Quantify the FoxJ1–positive or acetylated tubulin–positive cells as a percentage of total cells (DAPI–positive) per region of interest (ROI) using the cell counter Plugin in FIJI imaging software.
6. In the GraphPad Prism software (v. 10.0) generate graphs and perform statistical analysis using a two-tailed Student’s t-test after assessing the data distribution for normality using Shapiro-Wilk test.

6. Drug treatment of differentiated airway epithelia

1. At ALI day 21 prepare differentiation medium (3 mL for cells in three inserts) in a sterile tube at room temperature.
2. Add 7.5 µL of Diethylene triamine–NONOate or DETA/NO from a 100 mM stock solution (prepared in 10 mM NaOH) to the differentiation medium to achieve the final concentration of 250 µM in 3 mL. Mix gently by inversion and use immediately.
3. Add 750 µL of the final solution to each of the three basal chambers of differentiated cells and incubate for 24 h at 37 °C.
4. Prepare 1 mM Nω-Nitro-L-Arginine Methyl Ester (L–NAME) by adding 30 µL from the 100 mM L-NAME stock solution (prepared in DMSO) to MTEC differentiation media for a total volume of 3 mL.
5. Add 750 µL of the final solution to each of the three basal chambers and incubate for 24 h at 37 °C.
6. Add 30 µL of dimethyl sulfoxide (DMSO) only (untreated group) to 3 mL of basal media (1% v/v).
7. Add 750 µL of the final solution in the basal chamber of three inserts (untreated).

7. Sample collection

NOTE: For NOA analysis it is necessary to pool media, apical wash, and cell lysate samples from three inserts.

1. At ALI day 22, collect samples from the differentiating airway epithelia beginning with 750 µL of basal media from each of three inserts per experimental group.
2. Collect basal media incubated in inserts without cells to serve as blank control accounting for potential external nitrite contamination.
3. Pool the basal media from three inserts and store immediately at -20 °C until injection into NOA.
4. To collect the nitrite/NO present in the apical mucus of the epithelium, add 50 µL of pre-warmed PBS (1×) buffer (sterile, aliquoted and frozen) on top of cells in each insert and incubate for 30 min at 37 °C.
   NOTE: During the 30 min incubation of cells with PBS to collect apical wash, PBS can also be added in the basal chamber to maintain hydration on both sides of the cells.
5. Collect the PBS containing the mucus overlying the epithelium.
6. Pool the PBS washes from three inserts to obtain around 150 µL of apical mucus/PBS from each experimental condition which will be enough for two NOA injections.
7. Store the apical wash samples at -20 °C until further use.
8. Store also the PBS wash buffer for use as a blank control during NOA injections.
9. Wash the apical and basal sides of the cells in three inserts with 500 µL of prewarmed PBS twice.
10. Add 500 µL of Accutase solution apically and incubate for 15–20 min until the cells completely detach from the membranes.
11. Once fully detached, add serum-free RPMI-1640 media to the cells (at 3:1 media to Accutase ratio).
12. Pool the detached cells from the three inserts in a single tube.
13. Spin the cells at 3000 × g for 5 min at 4 °C.
14. Resuspend the cell pellet in 50 µL of lysis solution (RIPA buffer + Halt 1× protease inhibitor cocktail) and incubate on ice for 45 min.
15. Spin the lysate at 14,800 × g for 20 min at 4 °C and collect supernatant.
16. Use 3 µL of whole cell lysate to determine the protein concentration through the Bicinchoninic Acid (BCA) assay.
17. Dilute the remaining lysate solution 1:1 in RIPA buffer to make enough volume for two 50 µL NOA injections.
18. Store the lysates at -80 °C until injecting into the NOA at -20 °C.
19. Store the RIPA buffer used to resuspend the cell pellet at -20 °C to be used as blank and contamination control for lysate samples.

8. NOA calibration and sample injections

1. Prepare triiodide reagent by dissolving 400 mg of potassium iodide and 250 mg of iodine in 28 mL of glacial acetic acid and 8 mL of ultrapure water.
2. Perform the triiodide reagent preparation in a chemical fume hood using appropriate personal protective equipment (lab coat, gloves, and eye protection).
   CAUTION: Iodine and glacial acetic acid are hazardous and can cause irritation and corrosion to skin and eyes.
3. Store at room temperature and use within 2 weeks of preparation.
4. Prepare 1 mM stock solution of sodium nitrite in nitrite-free water.
5. Store the stock solution at -20 °C until use.
6. Prepare fresh working standards (0.125–4.0 µM) in PBS immediately before injections.
   NOTE: For NOA calibration, use sterile, tightly capped PBS to minimize nitrite contamination.
7. Turn on the nitrogen (N₂) carrier gas and initiate gas flow through the system.
8. Assemble the glass purge vessel and add 4 mL of triiodide reagent into the vessel with 20 µL of anti-foam diluted 1:10.
9. Securely cap it with a septum held in place by the screw cap to ensure an airtight seal.
10. Pour 10 mL of sodium hydroxide (NaOH) into the NaOH trap.
11. On the display screen of the NOA instrument (nCLD 88) select Measurement, Select Gas and Range.
12. Set the instrument to MR2:50.00, corresponding to a measurement range between 0 and 50 parts per billion of NO, and select Fast for filter setting.
13. Adjust the nitrogen gas pressure until the reactor pressure stabilizes between 27–29 psi (186–200 kPa).
14. Open the eDAQ chart software on the computer connected to the NOA.
15. Set the acquisition range to 10/s and the voltage to 10 V.
16. Navigate to Setup > Channel Settings, select Channel 1.
17. Set the display scale to 2:1 on the NOA software.
18. Start the data acquisition on the NOA instrument and in the software.
19. Monitor the baseline until it stabilizes.
20. Inject 10 µL of PBS through the septum into the purge vessel using a Hamilton syringe to confirm baseline stability.
21. Record the chemiluminescent signal for each injection.
22. Inject 10 µL of the 0.125 µM nitrite standard into the purge vessel.
23. Repeat twice for each nitrite standard, to obtain duplicate measurements that will later be averaged.
24. Annotate each injection in the Comment tab (e.g., 10 µL 0.5 µM nitrite).
25. Wipe the syringe on a lint-free tissue and rinse 3–4 times with sterile nitrite-free water to remove excess triiodide.
26. Repeat this cleaning step after each injection.
27. Allow the signal to return to baseline between injections (20–30 s).
28. Open the Flow Analysis window.
29. Review peak height and time values for each injection in the analysis table.
30. Highlight each peak and adjust the selection to calculate the area under the curve (AUC).
31. Toggle the display from S to C to enable calibration mode.
32. Enter the known nitrite amounts for each standard in the Amount column.
33. Click Calibration curve button to generate the standard curve.
34. Verify that the calibration curve meets acceptance criteria (R² ≥ 0.99).
35. Thaw biological samples previously collected from ALI cultures on ice.
36. Inject 50 µL of blank cell–free medium into purge vessel.
37. Inject 50 µL of each basal media sample into the purge vessel.
38. Record the chemiluminescence signal for each injection.
39. Perform duplicate injections (50 µL each) for each sample.
    NOTE: The 50 µL injections result in frequent bubble formation. To limit this issue, replace the triiodide reagent with anti–foam in the purge vessel regularly.
40. Annotate each sample in the Comment tab prior to injection.
41. Inject 50 µL of cell-free PBS (apical blank) into purge vessel.
42. Inject apical wash samples (two 50 µL injections per sample) and record the corresponding signal peaks.
43. Inject RIPA buffer twice, followed by lysate samples (two 50 µL injections per sample) and record the corresponding signal peaks.
    NOTE: Always allow the chemiluminescent trace to reach back to the baseline and stabilize. Wait 20–30 s, then inject the next sample.
44. At the end of sample injections, open the Flow Analysis window.
45. Highlight each sample peak to calculate the area under the curve (AUC).
46. Determine nitrite concentrations for each sample using the calibration curve generated from standard solutions.

9. Data collection, normalization and analysis

1. Export the data, including peak height (V), area under the curve (V.s) and mean area under the curve from the Flow Analysis function in the eDAQ software to an Excel file. The exported file will tabulate the height (V) versus time (s) of signal traces for the entire NOA experiment.
2. Determine the picomoles (pmol) of nitrite injected into the NOA by multiplying the nitrite concentration (µM) by the injection volume (µL).
3. Average the picomoles (pmol) of nitrite measured in controls (Cell-free media, PBS wash buffer or RIPA buffer).
4. Subtract the corresponding control values from the pmol measured in each 50 µL sample injection.
5. Determine the total picomoles (pmol) of nitrite in each sample by multiplying the pmol measured in the injected portion of the sample by the exact total sample volume collected. Use 750 µL for the media, 150 µL for the apical wash and around 90–100 µL for the lysate.
6. Additionally, for lysates only, multiply the total nitrite pmol by 2 to account for the dilution factor.
7. Normalize the pmol of nitrite obtained for each sample by dividing these values by the total protein amount (µg) present in the lysate of the corresponding sample, obtained previously through the BCA assay.
8. Average the nitrite levels (pmol/µg) from duplicate injections for each sample.
9. Plot normalized nitrite levels (pmol/µg of total protein) for each sample using GraphPad Prism software.
10. Assess data distribution for normality using the Shapiro-Wilk test.
11. Perform statistical analysis using a two-tailed Students’ t-test.

Results

The overall goal of this procedure is to quantify nitric oxide (NO) levels in mouse tracheal epithelial cell (MTEC) cultures differentiated at air–liquid interface (ALI). Primary cells are isolated and differentiated according to established protocols^27,28. Over the course of 21 days at ALI, the cells are differentiated into a pseudostratified airway epithelium containing ciliated cells^27,28. Levels of ci...

Discussion

Multi–ciliated cells within the airway epithelium are critical for NO synthesis and signaling, which in turn is essential for regulating ciliary beating, mucociliary clearance, and airway homeostasis^6,7,8. NO is a highly reactive molecule and even small changes in NO levels are associated with airway diseases^1,2,3,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4′,6-Diamidino-2-phenylindole (DAPI)	ThermoFisher Scientific	62248	diluted 1:2000 for immunostaining
50 mL Centrifuge tube filter (0.22 µm)	CellTreat	229710	For preparation of sterile solutions
500 mL Filter units	ThermoFisher Scientific	566-0020	For sterile media preparation
Accutase	Sigma	A6964
Amphoterecin B	ThermoFisher Scientific	15290026
Bovine Pituitary Extract	ThermoFisher Scientific	13028014
Bovine Serum Albumin	Sigma	A7906
Cholera toxin	Sigma	C8052
Collagen I	ThermoFisher Scientific	A1048301
Confocal Microscope	Nikon		W1 Spinning disk Ti2 inverted microscope with Hamamatsu sCMOS camera
Corning Transwell inserts	Sigma	CLS3460
Diethylenetriamine NONOate (DETA-NONOate)	Cayman Chemical	82120
DMEM/F-12 media	ThermoFisher Scientific	11330032
DNAse I	Sigma	DN25
Fetal Bovine Serum	Sigma	F4135
Halt 1X Protease inhibitor cocktail	ThermoFisher Scientific	1860932
Ham's F1-12 Nutrient mix	ThermoFisher Scientific	11765054
Insulin solution	Sigma	I0516
Isoproterenol hydrochloride	Sigma	I6504
Keratinocyte Serum-free media	ThermoFisher Scientific	17005042
Murine Epidermal growth factor	Sigma	E4127
N(G)-Nitro-L-arginine methyl ester (L-NAME)	Sigma	N5751
NuSerum	Corning	355100
Penicillin-Streptomycin	Sigma	P4333
Phosphate buffer saline (PBS)	ThermoFisher Scientific	10010023
Pierce BCA Protein Assay Kit	ThermoFisher Scientific	23227
Pronase	Sigma	10165921001
Retinoic acid	Sigma	R2625
Rho kinase inhibitor (Y-27632 hydrochloride)	Cayman Chemical	TOM-C837Z37
RIPA buffer	Sigma	R0278
RPMI-1640 media	ThermoFisher Scientific	11875093
Sodium nitrite	Sigma	S2252
Transferrin	Sigma	T8158
α-Acetylated tubulin antibody (mouse)	Sigma	T6793	diluted 1:200 for immunostaining
α-FoxJ1 antibody (mouse)	ThermoFisher Scientific	14-9965-80	diluted 1:200 for immunostaining
α-mouseCy3 secondary antibody	ThermoFisher Scientific	A10521	diluted 1:1000 for immunostaining
γ-secretase inhibitor IX (DAPT)	Sigma	D5942