Home › Cell Biology › Application of Genetically Encoded Fluorescent Nitric Oxide (NO•) Probes, the geNOps, for Real-time Imaging of NO• Signals in Single Cells
Cell Biology JoVE (Open Access) Citable · DOI

Application of Genetically Encoded Fluorescent Nitric Oxide (NO•) Probes, the geNOps, for Real-time Imaging of NO• Signals in Single Cells

DOI: 10.3791/55486-v
What you'll learn
  • ✓ Apply geNOps probes to monitor NO• dynamics in single endothelial cells
  • ✓ Perform simultaneous multichannel fluorescence imaging of NO• and Ca2+ signals
  • ✓ Interpret real-time NO• fluctuations triggered by calcium signaling events
Protocol

This manuscript presents protocols for the application of novel genetically encoded nitric oxide (NO•) probes (geNOps) to monitor single cell NO• fluctuations in real-time using fluorescence microscopy. The Ca2+-triggered NO• formation on the level of individual endothelial cells was visualized by combining geNOps with a chemical Ca2+ sensor.

Difficulty
advanced
Total time
~3–4 hours (cell preparation + live imaging per sample)
Model organism
EA.hy926 (human endothelial cell line)
Biosafety
BSL-1

Steps

1
Prepare endothelial cells for imaging

Culture and prepare EA.hy926 endothelial cells for live-cell imaging. Ensure cells are healthy and adherent on appropriate imaging substrates.

▶ 01:07
2
Image NO• and Ca2+ signals simultaneously

Set up multi-channel fluorescence microscopy using geNOps to detect NO• and a chemical Ca2+ sensor. Perform real-time imaging of individual cells under physiological stimulation conditions.

▶ 02:33
3
Analyze multichannel signals in single cells

Collect and evaluate simultaneous NO• and Ca2+ fluorescence data from individual endothelial cells. Correlate calcium-triggered NO• formation with temporal dynamics.

▶ 06:28
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