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Method Article

Fluorescence-Based Calcium Imaging in Primary Human Airway Epithelial Cultures Using Automated Cell Segmentation

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DOI:

10.3791/69470

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May 22nd, 2026

 ,  , 
1Molecular Medicine, Hospital for Sick Children, 2Department of Biochemistry, University of Toronto, 3Department of Physiology, University of Toronto

In This Article

Summary

Live-cell imaging of calcium signaling is utilized extensively to study calcium mobilization in stable cell lines grown in monolayers. This protocol describes live-cell fluorescence imaging of calcium signaling using live-cell dyes on complex, primary tissue, and also utilizes machine learning software to quantify the fluorescence intensity of thousands of individual cells simultaneously, thereby streamlining analyses.

Abstract

Calcium signaling is critical in a multitude of biological processes, reinforcing the need to develop methods to study calcium flux in primary tissue. The study of calcium signaling at a single-cell resolution in complex primary epithelial cultures is challenging and therefore remains poorly studied. This study presents methods adapted for monitoring live calcium signaling in primary airway epithelial cultures and novel approaches for machine learning based analyses. Using patient-derived primary airway epithelial cultures differentiated at ALI (air-liquid interface) loaded with an extrinsic fluorescent indicator dye, calcium mobilization was measured at single-cell resolution using an epifluorescent microscope. We developed a novel software that utilizes machine learning-based cell segmentation to assign fluorescence intensities to distinct cells over time. Overall, the adapted imaging setup and software enable a rapid and unbiased approach to analyzing single cells within the primary epithelial cultures. The step-by-step protocol presented here will enable the future study of calcium in individual cells and cell types that make up the primary epithelial cultures.

Introduction

Changes in intracellular calcium mobilization dictate a wide variety of cellular processes, including changes in gene expression and modulation of innate immune responses1,2. Intracellular calcium levels can be altered through release from intracellular calcium stores, such as the endoplasmic reticulum and mitochondria1,2,3. Extracellular calcium influx into the intracellular space can also alter cytosolic calcium through both passive and active transport2. Live cell imaging of calcium mobilization is assayed primarily by intracellular dyes or genetically encoded dyes, GCaMP (Green Calcium-activated Multimer/Protein)4. Common in literature are AM (acetoxymethyl) conjugated dyes, which enable the dye to permeate the plasma membrane5,6,7,8. Upon entering the cell, the AM moiety is cleaved by endogenous intracellular esterases, and the dye gains a negative charge, which traps it in the cytosolic space9. Due to this gained charge, the dye can be pumped out by plasma membrane resident organic anion channels. Therefore, probenecid, an inhibitor of organic anion efflux transporters, is added to prevent cellular efflux of the dye and retain optimal cytosolic signal10,11. Studies commonly employ Fura-2 AM, a ratiometric dye, and Fluo-4AM, an intensitometric dye, to assay calcium signaling on stable cell lines cultured in a monolayer. This study employs a protocol adapted for epifluorescent imaging of multilayered cultures grown at the air-liquid interface using the recently developed intensitometric calcium dye, Cal-520 AM, which has enhanced sensitivity compared to Fluo-4 AM. Thus, it is suitable to robustly visualize subtle signals at a single cell resolution from complex primary epithelial cultures. Current literature is rich in live calcium imaging of bulk responses from stable cell lines. However, it is now commonly known that in vivo, tissues constitute a diverse cellular landscape supporting different cell type-specific roles in maintaining tissue homeostasis12,13,14,15. Despite this common knowledge, the mechanisms by which distinct cell types coordinate calcium signals and regulate calcium ion homeostasis remain unknown. Therefore, the methods presented here will enable single-cell detection of calcium signaling and enhance the throughput of downstream analyses in primary airway tissues.

We are leveraging our previously published and rigorously validated primary epithelial culture models of the human nasal epithelium as a tool to image live cell, cytosolic calcium in a more biologically relevant model16,17,18. Briefly, epithelial cells are isolated from patient nasal brushes and are cultured on a semipermeable membrane on a transwell, which is suspended within a culture well in order to maintain an air-liquid interface (ALI). This interface, coupled with specialized media, has been rigorously tested using orthogonal techniques for the presence of different cell types16,17,18. Moreover, it has been shown that the current methods are translatable to primary bronchial cultures that are cultured in a similar way to our primary nasal cultures19.

This article introduces an adapted method to measure and analyze live calcium signaling at a single-cell resolution in these primary airway epithelial cultures. These multilayered cultures are difficult to visualize using microscopy; however, the current setup enables us to focus reliably on single layers within the multilayered cultures, allowing us to detect calcium mobilization from these specific layers. This setup has several advantages, in that it does not require a perfusion system, which is often costly and difficult to adapt unless specialized microscopes are employed. Epifluorescence microscopy has been employed, which offers improved temporal resolution compared to confocal imaging. The use of light-emitting diodes (LEDs) minimizes phototoxicity relative to laser-based confocal systems. Extrinsic intensiometric dyes are used in the protocol presented here as they enable rapid, relative changes in calcium across the entire cell population and are compatible with most fluorescence microscopy setups. This is beneficial over common ratiometric dyes, which require deep ultraviolet (UV) excitation that is not available on many microscopes. Moreover, GCaMP requires transfection or transduction, which strengthens the technique for more targeted calcium imaging in specific cell types. Due to low transduction and transfection efficiencies in primary epithelial cultures, the technique is poorly suited for monitoring the entire cell population.

Additionally, this study developed novel, specialized machine learning based software that allows single-cell segmentation based on live nuclear staining. The software is then able to record fluorescence intensity values for each cell over time. A stable epithelial cell line (Calu-3) was initially employed for method optimization. Then, these methods were translated to study single-cell calcium mobilization in primary epithelial cultures. Variables such as concentration of the calcium dye and probenecid, timing of incubation steps, as well as the imaging setup were optimized. Here, calcium signaling is recorded at a frame rate of 0.2 fps (frames per second), which is suitable for monitoring calcium signaling. The frame rate can be increased in order to measure more rapid calcium transients20. The optimal variables were then applied and further optimized in more complicated primary nasal cultures.

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Protocol

The reagents and the equipment used are listed in the Table of Materials.

1. Calu-3 cell maintenance and culturing

  1. Maintain Calu-3 cells in T-25 flasks in EMEM culture media supplemented with 20% FBS and 1% penicillin/streptomycin. Prewarm the media and change it on alternating days (5 mL/flask).
  2. When cells are 70% confluent in the flask, aspirate the media in the flask.
  3. Wash the flask with 5 mL 1x phosphate-buffered saline (PBS).
  4. Add 1 mL of TrypLE directly to the cells for 10-15 min at 37 °C, 5% CO2. Ensure that all cells have detached from the bottom of the flask and are dissociated into a single-cell suspension.
  5. Neutralize the reaction with 4 mL of fresh culture media (step 1.1), then mix the cells well with a serological pipette to ensure even seeding.
  6. Plate the cells onto a 96-well plate at 90%-100% confluence (approximately 40,000 cells/well).
    1. To seed wells, add 200 µL/well of well-mixed cell suspension.
    2. Allow the cells to adhere to the 96-well plate overnight.
  7. Change media every day (200 µL/well) until ready for experiments.
  8. Allow 2-5 days post confluency within wells to start experimentation prior to conducting experiments to allow for differentiation.

2. Protocol for loading Calu-3 cells with Cal 520 AM or Fluo-4 AM

CAUTION: Fluo-4 AM and Cal-520 AM are dissolved in DMSO, which is considered hazardous. Probenecid is listed as an acute hazard. Please wear gloves and a lab coat when handling chemicals to avoid contact with skin. Dispose of all chemicals into designated chemical waste bins.

  1. On the day of the experiment, remove media from the wells by pipetting and wash cells twice (200 µL/well) in prewarmed calcium buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl2 2 mM CaCl2, and 10 mM Hepes, adjusted to pH 7.38 (NaOH) 291 mOsm), to remove dead cells.
  2. Incubate cells with 200 µL/well of a cocktail of 3 µM Cal-520 AM, 2.5 mM probenecid, and 1:5000 Hoechst nuclear stain in calcium buffer (step 2.1) for 1 h in an incubator set at 37°C and 5% CO2.
    1. For Fluo-4 AM experiments, incubate cells with 200 µL/well of a cocktail of 3 µM Fluo-4 AM, 2.5 mM probenecid, and 1:5000 Hoechst nuclear stain in calcium buffer (step 2.1) for 1 h at 37 °C and 5% CO2.
      NOTE: Keep the tubes of dye in the dark by covering them with aluminum foil.
  3. During the incubation, prepare thapsigargin in calcium buffer (step 2.1). Adjust intermediate stock concentrations to ensure proper final concentrations within the well.
    NOTE: Here, 50 µL of drug is added to 100 µL of the existing calcium buffer. Therefore, a 3x intermediate stock of 6 µM was prepared.
  4. After 1 h, aspirate the dye by pipetting and wash away extracellular dye with calcium buffer (step 2.1) twice (200 µL/well).
  5. Following washes, add 100 µL/well of 2.5 mM probenecid diluted in calcium buffer (step 2.1) to maintain the dye in the cytoplasmic space.
  6. Wrap the plate in aluminum foil to protect it from light. The cells are now ready to image.

3. Setting up microscopy for the Calu-3 stable cell line

  1. Using the epifluorescence microscope equipped with a SuperFluor Objective, a stable, solid-state light source, and a CMOS fluorescence camera (6.5 µm pixel size). Illumination intensity is set at 500 mW.
  2. Place the plate of cells on the stage using the 96-well plate adapter. Using Brightfield, focus on the cell layer at a low magnification to find the monolayer of cells (use 5-10x air objective).
    NOTE: This step is optional, but helpful to gain initial focus on the cell layer efficiently.
  3. Once the cell layer has been identified, use Brightfield and switch to a higher magnification (20x air objective) and refocus on the cell layer.
    NOTE: Ensure proper focus on the monolayer with Brightfield; cell boundaries should be clear.
  4. Identify the calcium dye signal using the GFP (475 nm LED). Ensure that the LED power falls between 5%-10% to avoid photobleaching, and the exposure time is suitable to visualize baseline green signal without oversaturating the signal, typically between 300-500 ms.
    NOTE: To ensure reproducibility and even dye loading, qualitatively analyze the cell layer; most cells should exhibit the same brightness. Focus on fields of view with limited dead cells, which will fluoresce intensely before any stimulant is added. Relative baseline fluorescence intensity can be measured using Fiji (step 7.9). Baseline fluorescence variation between cells should be close to 10%-15% if dye has been loaded evenly (step 7.9).
  5. Identify the Hoechst nuclear stain using the DAPI (405 nm) LED. LED power should be between 2%-5% with an exposure time between 50-100 ms.
    NOTE: If the cells are in focus, nuclei should appear with sharp outlines. Ensure that the nuclear signal does not reach saturation, as nuclei can interfere with the green channel.

4. Acquiring live calcium video

  1. Start a live video using an interval of 1 frame every 5 s (0.2 fps) for a duration of 10 min.
    NOTE: Capture each fluorescent channel (DAPI 405 nm and GFP 475 nm) at each frame.
  2. Once the video has started, allow a 5 min baseline reading prior to adding agonists.
  3. Using a P200 pipette, add 50 µL of thapsigargin at a 3x intermediate stock to the well containing 100 µL of 2.5 mM probenecid in calcium buffer (step 2.1). Gently resuspend 1-2 times within the well to ensure the drug is evenly dispersed. Continue reading for 5 min following the addition of thapsigargin.
    NOTE: The drug addition step is critical; the pipette tip should not contact the plate to shift the plane of focus. Instead, float the pipette tip to only contact the buffer within the well. If drugs are mixed too vigorously, cells can detach from the plate and interfere with the signal.
    NOTE: Ensure to take note of the frame in which the drug is added for downstream analysis.
  4. Save the video and the snapshot taken prior to the experiment. Proceed to data analysis.

5. Protocol for loading primary airway cells with Cal 520 AM or Fluo-4 AM

  1. On the day of the experiment, remove basolateral media from the trans well and wash the apical and basolateral sides of the insert twice using 750 µL basolateral and 300 µL apical of the calcium buffer (step 2.1).
  2. Incubate the cells with 600 µL basolateral and 200 µL apical of the Cal-520 AM or Fluo-4 AM dyes for 1 h at 37 °C and 5% CO2. Prepare the dye in the same way as for the Calu-3 cells, for Cal-520 AM, 3 µM Cal-520 AM, 2.5 mM probenecid, and 1:5000 Hoechst in calcium buffer (step 2.1). For Fluo-4 AM, 3 µM Fluo-4 AM, 2.5 mM probenecid, and 1:5000 Hoechst in the same calcium buffer.
  3. During the incubation period, prepare thapsigargin at a 3× intermediate stock for a final concentration of 2 µM (6 µM intermediate stock).
  4. Following this, aspirate the dye and wash the cells twice with 300 µL apical and 750 µL basolateral buffer containing calcium (step 2.1).
  5. Following washes, add 100 µL apical and 500 µL basolateral of 2.5 mM probenecid in calcium buffer (step 2.1).
  6. Keeping the trans well within the plate, wrap the plate in foil. The cells are now ready for imaging.

6. Setting up microscopy for the primary nasal inserts

  1. Acquire a glass-bottom Petri dish suitable for imaging, tweezers, and thin masking tape. Ensure that the Petri dish is clean by quickly wiping it with 70% ethanol and allowing it to dry completely.
  2. Place the glass-bottom Petri dish within the small circular adaptor for the epifluorescence microscope.
  3. Using tweezers, carefully place the insert into the center of the glass-bottom dish.
    NOTE: Be careful not to spill the buffer, which is placed in the apical compartment of the insert.
  4. To ensure the insert doesn't move during drug additions, use masking tape to secure the top of the plastic of the insert onto the sides of the glass-bottom dish.
  5. Place the adapter with the insert attached onto the stage of the microscope and begin by focusing on the cells using the 10x air brightfield objective.
  6. Identify the calcium dye signal using the GFP (475nm LED). Ensure that the LED power falls between 5%-10% to avoid photobleaching, and the exposure time is suitable to visualize the baseline green signal without oversaturating it, typically between 300-500 ms.
    NOTE: Please see the NOTE under step 3.4 to ensure proper calcium dye loading.
  7. Identify the Hoechst nuclear stain using the DAPI (405 nm) LED. LED power should be between 2%-5% with an exposure time between 50-100 ms.
    NOTE: If the cells are in focus, nuclei should appear with clear, defined outlines. Focus on the most apical layer of cells. Adjust the microscope's z-axis to identify the highest focal plane where cells remain in sharp focus. Ensure that the nuclei do not reach saturation of signal, as the nuclear signal can interfere with the green channel.
  8. To acquire the live calcium video, follow steps 5.1-5.4. During the drug addition step, add into the center of the insert and resuspend slowly using a P200 pipette.
    NOTE: Ensure to take note of the frame in which the drug is added for downstream analysis.

7. Analysis using the Fiji software

  1. Open the Fiji application and Select File \ Open, upload the video file, and on the resulting pop-up window, keep all default settings.
  2. If desired, under Image \ Adjust \ Brightness/Contrast, alter the brightness and contrast of the separate fluorescence channels to have the most optimal view of the cells.
  3. To ease cell tracing, merge the nuclear stain or brightfield channels using Image \ Colour \ Merge Channels and select the appropriate files for each individual channel in the pop-up.
  4. To trace specific cells based on cell boundaries and nuclear stain, use Analyze \ Tools \ ROI Manager and check off both show all and labels in the resulting pop-up.
  5. Carefully trace cells using the freehand selections tool and press t to add a cell.
    NOTE: Ensure that cells are traced in all 4 corners and the center of the video.
  6. Once all cells have been traced accurately, within the open tab with all ROIs, click on More \ Multi Measure, and press OK on the resulting pop-up.
  7. Copy the entire sheet in the resulting pop-up entitled Results into an excel file.
    NOTE: The resulting sheet will have 6 subheadings (Area1, Mean1, Min1, Max1, IntDen1, RawIntDen1) for each ROI with a numerical value after each, which corresponds to the respective cell that was traced. The important value for downstream analysis will be the Mean of each ROI.
  8. In order to monitor the variation between fluorescence intensity, calculate the average fluorescence during baseline for each cell, and calculate the percent variance. Experiments typically have between 20%-30% variance.
    Coefficient of variation formula, static equation, educational research use.
  9. Normalize all readings by dividing the mean intensity values by the first reading of baseline for each of the cells traced.
  10. Create an additional column for time in seconds, with the first frame corresponding to frame 0 and subsequent timepoints increasing by 5, which corresponds to the 0.2 fps interval.
  11. Copy the data into an analysis and graphing software and generate a curve of change in fluorescence over baseline over time.
    NOTE: To generate single-cell maxima plots, use the =MAX() function in Excel to find the frame at which maximal fluorescence intensity is reached for each cell and plot using GraphPad Prism.

8. Analysis using the Calcium Suite

  1. Using Fiji, open the video file and follow steps 8.1 and 8.2.
  2. Once the nuclear and green channels are optimized for viewing, duplicate a single frame of the video by clicking on Image \ Duplicate. In the resulting pop-up, deselect Duplicate Hyper stack. Within the same pop-up, under channels, type 1. Save it as a PNG image file.
  3. Repeat step 9.2 and under channels, type 2.
    Merge the two images by following step 8.3. Upload the image into the Cell-Pose GUI (Graphical User Interface) to generate a cell mask.
  4. Upload the image into the Cell-pose GUI, and segment using the nuclear stain (Channel 1) and cytoplasmic stains (Channel 2) by selecting Cyto3 model.
  5. Save the image as a PNG file, then open the PNG file in Fiji and convert it to a TIFF file.
    NOTE: Cell-pose GUI produces a black-and-white PNG file.
  6. Open both the mask file and the video file in the Calcium Suite by dragging and dropping, and then click on Analyze Data to generate a downloadable plot, as well as a downloadable .csv file containing normalized and raw intensity values for all cells counted by the software.

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Results

To measure calcium mobilization in live cells, Calu-3 cells or differentiated primary nasal cultures are incubated with an intracellular dye. The relative change in calcium-dependent signal is measured as an increase in the mobilization of calcium either from intracellular calcium stores or the extracellular space into the cytosol. A sustained increase in calcium into the cytosol is represented by an increase in fluorescence after thapsigargin, a well-characterized irreversible inhibitor of the ER-resident SERCA (Sarco/E...

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Discussion

This article has presented methods for analyzing single-cell calcium signaling in live primary nasal and bronchial cultures as well as a stable epithelial cell line. Both the Cal-520 AM and Fluo-4 AM calcium reporters are useful calcium indicators in both setups. Cal-520 AM is more sensitive as it has a higher quantum yield of 0.75 compared to Fluo-4 AM of 0.16 and slightly higher affinity for calcium (The Kd of Cal-520 AM is 320 nM and Fluo-4 AM is 345 nM). Moreover, Cal-520 AM is reported as having improved cellular re...

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Disclosures

The authors declare no competing interests.

Acknowledgements

The authors would like to acknowledge the assistance of culturing the primary nasal tissue by Tarini Gunawardena. As well as Wu Shu from the University of Iowa for generously culturing and shipping the primary bronchial cultures. As well as Gabrielle Langeveld and Dian Liu for help with analysis and designing imaging adapters. We would also like to thank the Imaging Facility at the Hospital for Sick Children. We also acknowledge funding by Cystic Fibrosis Canada and the US Cystic Fibrosis Foundation.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
24-well plate Thermofisher 142475
24-well transwell inserts Corning CLS3470-48EA
96-well Flat Clear Bottom Black Polystyrene TC-treated MicroplatesCorning 3603
Cal-520 AMAAT Bioquest 21130Aliquoted and stored at -20°C, dissolved in DMSO
Calcium Suite SoftwareThe code can be requested by contacting our laboratory group
EMEM, 1xsWisent 320-005-CLStored at 4°C 
Fetal Bovine Serum Wisent 080-450Aliquoted and stored at -20°C
Fluo-4 AM AAT Bioquest 20550Aliquoted and stored at -20°C, dissolved in DMSO
HEPESBioshopHEP001.5Stored at Room Temperature
Hoechst Thermofisher H3570Stored at 4°C 
Human Epithelial Colorectal Adeoncarcinoma Cell Line ATCCHTB-55
Magnesium Chloride Sigma 7786-30-3Stored at Room Temperature
micro-dish 35mm, low glass bottomIbidi 80137Stored at Room Temperature 
Penicillin-Streptomycin SolutionWisent 450-200-ELStored at 4°C
Phosphate-Buffered Saline (PBS)Wisent 3111-010-CLStored at Room Temperature 
Potassium Chloride Sigma 7447-40-7Stored at Room Temperature 
Probenecid ThermofisherP36400Aliquoted and stored at -20°C , water soluble 
Sodium Chloride Bioshop SOD004Stored at Room Temperature 
Thapsigargin ThermofisherT7458Aliquoted and stored at -20°C Dissolved in DMSO 
TripLE Express Enzyme (1X), phenol red Thermofisher12605010Stored at 4°C

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Fluorescence MicroscopyCalcium SignalingMachine Learning AnalysisSingle Cell ResolutionThapsigargin StimulationFluorescent Indicator DyeCell Mask Generation
Fluorescence-Based Calcium Imaging in Primary Human Airway Epithelial Cultures Using Automated Cell Segmentation
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