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

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 responses^1,2. Intracellular calcium levels can be altered through release from intracellular calcium stores, such as the endoplasmic reticulum and mitochondria^1,2,3. Extracellular calcium influx into the intracellular space can also alter cytosolic calcium through both passive and active transport². Live cell imaging of calcium mobiliza....

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 o....

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.......

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
24-well plate	Thermofisher	142475
24-well transwell inserts	Corning	CLS3470-48EA
96-well Flat Clear Bottom Black Polystyrene TC-treated Microplates	Corning	3603
Cal-520 AM	AAT Bioquest	21130	Aliquoted and stored at -20°C, dissolved in DMSO
Calcium Suite Software	The code can be requested by contacting our laboratory group
EMEM, 1xs	Wisent	320-005-CL	Stored at 4°C
Fetal Bovine Serum	Wisent	080-450	Aliquoted and stored at -20°C
Fluo-4 AM	AAT Bioquest	20550	Aliquoted and stored at -20°C, dissolved in DMSO
HEPES	Bioshop	HEP001.5	Stored at Room Temperature
Hoechst	Thermofisher	H3570	Stored at 4°C
Human Epithelial Colorectal Adeoncarcinoma Cell Line	ATCC	HTB-55
Magnesium Chloride	Sigma	7786-30-3	Stored at Room Temperature
micro-dish 35mm, low glass bottom	Ibidi	80137	Stored at Room Temperature
Penicillin-Streptomycin Solution	Wisent	450-200-EL	Stored at 4°C
Phosphate-Buffered Saline (PBS)	Wisent	3111-010-CL	Stored at Room Temperature
Potassium Chloride	Sigma	7447-40-7	Stored at Room Temperature
Probenecid	Thermofisher	P36400	Aliquoted and stored at -20°C , water soluble
Sodium Chloride	Bioshop	SOD004	Stored at Room Temperature
Thapsigargin	Thermofisher	T7458	Aliquoted and stored at -20°C Dissolved in DMSO
TripLE Express Enzyme (1X), phenol red	Thermofisher	12605010	Stored at 4°C