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

Single-cell transcriptomics reveal heterogeneity in cystic fibrosis airway epithelium. We intend to understand how cell type-specific functions and changes affect disease pathogenesis. The current challenge is to accurately measure single-cell proteomics and functions.

This still requires technical development. To begin, maintain Calu-3 cells in T-25 flasks containing Eagle's Minimum Essential Medium Culture Media supplemented with 20%FBS and 1%penicillin streptomycin. When the cells reach approximately 70%confluence, wash the cells with five milliliters of PBS.

After washing, add one milliliter of TrypLE directly onto the cells and place the flask in an incubator set to 37 degrees Celsius and 5%carbon dioxide for 10 to 15 minutes. Then neutralize the TrypLE by adding four milliliters of fresh complete culture media and mix the cell suspension thoroughly. Plate the cells into a 96-well plate at approximately 90%to 100%confluence, corresponding to about 40, 000 cells per well.

Allow the cells to adhere to the 96-well plate overnight. Change the media every day and continue culturing the cells for two to five days post-confluency to allow for differentiation. On the experiment day, wash the Calu-3 cells and incubate them with appropriate dyes.

Next, add 100 microliters per well of 2.5 millimolar probenecid diluted in calcium buffer to maintain the dye within the cytoplasmic space and wrap the plate in aluminum foil to protect it from light until imaging. For primary airway cells. Remove the basolateral media from the transwell and wash the apical side by adding 300 microliters of calcium buffer.

Then wash the basolateral side by adding 750 microliters of calcium buffer. Remove the buffer by pipetting and repeat the wash once more on both sides. Next, incubate the cells with 200 microliters of dye solution on the apical side and 600 microliters on the basolateral side for one hour at 37 degrees Celsius and 5%carbon dioxide.

During the incubation, prepare thapsigargin diluted in calcium buffer as a three times intermediate stock. Adjust the concentration to six micromolar to achieve a final concentration of two micromolar in the well. After incubation, remove the dye solution from both apical and basolateral compartments by pipetting.

Wash the cells twice with 300 microliters of calcium buffer to the apical side and 750 microliters of calcium buffer to the basolateral side. Then remove the buffer by pipetting. Next, add 100 microliters of 2.5 millimolar probenecid in calcium buffer to the apical compartment, and add 500 microliters of the same solution to the basolateral compartment.

Keeping the transwell insert within the plate, wrap the entire plate in aluminum foil to protect it from light, and set the wrapped plate aside for imaging. Open the Fiji application. Select File and choose Open to upload the video file.

In the resulting pop-up window, keep all default settings and confirm the selection. If desired, adjust the image display by selecting Image, choosing Adjust, and clicking Brightness Contrast. Modify the brightness and contrast of individual fluorescence channels to achieve an optimal view of the cells.

To ease cell tracing, merge channels by selecting Image, choosing Color, and clicking Merge Channels. In the pop-up window, assign the appropriate files to each individual channel and confirm the selection. For tracing, select Analyze, Choose Tools, and open ROI Manager.

In the resulting pop-up window, enable both Show All and Labels options. Using the freehand selections tool, carefully trace individual cells based on cell boundaries and the nuclear stain. Then press the T key to add each traced cell to the ROI list.

Ensure that cells are traced in all four corners of the field of view and in the center of the video. Once all cells have been accurately traced, within the ROI Manager window, click More and select Multi Measure. In the resulting popup window, press OK to begin the measurement.

Open the video file in Fiji. Optimize the nuclear and green fluorescence channels for viewing using the image adjustment tools. Once the channels are optimized, duplicate a single frame by selecting Image and choosing Duplicate.

In the resulting pop-up window, deselect Duplicate stack and save the duplicated image as a PNG image file. Repeat the frame duplication for the other channel by selecting Image and choosing Duplicate. In the popup window, press OK, and save the image is a PNG file.

Merge the two saved images by selecting Image, choosing Color, and clicking Merge Channels. Then assign each PNG file to the appropriate channel and confirm the merge. Upload the merged image into the cell post-graphical user interface to generate a cell mask.

Segment the image using the nuclear stain as channel one and the cytoplasmic stain is channel two by selecting the cyto3 model. Now, save the generated mask image as a PNG file. Open it in Fiji and convert it to a TIFF file.

Finally open both the mask file and the original video file in the calcium suite by dragging and dropping them into the software interface. Click Analyze Data to generate a downloadable plot and a downloadable CSV file containing normalized and raw intensity values for all analyzed cells. Fluorescence intensity normalized to baseline increased over time in individual Calu-3 cells following thapsigargin stimulation when measured using both Fluo-4 AM and Cal-520 AM, the maximal calcium-dependent fluorescence activation after thapsigargin was significantly higher in Calu-3 cells measured with Cal-520 AM compared to Fluo-4 AM.Similarly, individual primary airway and bronchial cells showed increased normalized fluorescence over time after thapsigargin stimulation using either of the dyes.

Additionally, maximal calcium-dependent fluorescence activation was significantly higher in cells loaded with Cal-520 AM.Machine learning-based analysis identified and quantified calcium-dependent fluorescence changes in 1, 441 individual cells, from a single field of view. Normalized fluorescence traces generated by the software showed calcium responses for all 1, 441 identified cells following thapsigargin stimulation. Our studies will help in understanding distinct cell types'responses to their environment, which will aid in the development of targeted therapeutics.

Our findings have the potential to zero in on distinct cell types, to assign certain live cell responses to environmental stimuli. In the future, we intend to explore how cell type-specific signaling in disease and healthy cells is altered to determine the types that are responsible.