Home › Cell Biology › Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells
Cell Biology JoVE (Open Access) Citable · DOI

Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells

DOI: 10.3791/59815-v
What you'll learn
  • ✓ Measure airway surface liquid pH dynamically in primary human airway epithelial cells
  • ✓ Perform in situ pH calibration using fluorescent plate-reader methodology
  • ✓ Analyze real-time kinetics data from thin-film airway epithelial cultures
Protocol

We present a protocol to make dynamic measurements of the airway surface liquid pH under thin film conditions using a plate-reader.

Difficulty
advanced
Total time
~4–6 hours per experiment (including calibration and kinetics measurements)
Model organism
Primary human airway epithelial cells (hAEC)
Biosafety
BSL-1

Steps

1
Prepare primary human airway epithelial cells

Culture and prepare hAECs for fluorescent pH measurement under thin-film conditions. Ensure cells are viable and properly seeded in plate-reader compatible format.

▶ 01:09
2
Acquire background fluorescence measurements

Establish baseline fluorescent signal from cell monolayers without pH-sensitive dye or probe interference. Record background readings to subtract from kinetic measurements.

▶ 01:53
3
Measure airway surface liquid pH kinetics

Conduct real-time fluorescent measurement of pH changes in airway surface liquid during the experimental protocol using the plate-reader in kinetics mode.

▶ 04:43
4
Perform in situ pH calibration procedure

Calibrate fluorescent signal using known pH buffers applied directly to cell cultures to establish a standard curve for pH determination.

▶ 05:45
5
Analyze fluorescent data and calculate pH

Process raw kinetics data, apply calibration curve, and calculate airway surface liquid pH values from fluorescent intensity measurements.

▶ 06:28
6
Interpret representative pH calibration results

Review and evaluate representative pH calibration outcomes from primary hAECs to validate measurement accuracy and protocol reproducibility.

▶ 09:34
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