Detecting Hydrogen Peroxide Levels in Cultured Neurons Through Live-Cell Fluorescence Imaging

Abstract

Source: Terzi, A. et al., ROS Live Cell Imaging During Neuronal Development. J. Vis. Exp. (2021).

This video demonstrates a technique to detect real-time hydrogen peroxide levels in retinal ganglion cells using the roGFP2-Orp1 biosensor and live cell imaging techniques. It outlines the steps involved in cell preparation, fluorescence imaging, and the quantification of hydrogen peroxide levels.

Protocol

1. In vitro reactive oxygen species (ROS) imaging of cultured retinal ganglion cell (RGC) neurons

1. On the day of imaging (typically 6-24 h after cell plating), check retinal ganglion cells under a microscope to validate the growth of RGC axons.
2. For live-cell imaging, transfer coverslips from the culture dish to a live cell imaging chamber.
3. Set up microscope for imaging. Use an inverted microscope equipped with a differential interference contrast (DIC) objective, OG590 long-pass red filter, and an EM-CCD camera.
4. Before imaging, replace the zebrafish cell culture medium with serum and antibiotics (ZFCM)(+)) with zebrafish cell culture medium without serum and antibiotics (ZFCM(-)).
5. Once cells are positioned with 10x objective, acquire images at 60x magnification using a high NA oil immersion objective. Use an additional 1.5x magnification.
6. First, acquire DIC images. Then, image roGFP2-Orp1 using an appropriate filter set. Excite roGFP2-Orp1 with 405/20 and 480/30 nm excitation filters sequentially and acquire images with 535/30 nm emission filter after emission light has passed the dichroic mirror 505DCXR.
7. After taking the first set of images, exchange media with media containing different treatment solutions. The media should be changed every 30 minutes of imaging to avoid pH and osmolarity changes.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
35-mm culture dish	Sarstedt	83-3900
Cover glass	Corning	2850-22
Disposable Petri Dishes (100 x 15 mm)	VWR	25384-094
Fetal Bovine Serum	ThermoFisher Scientific	26140087
Inverted Microscope	Nikon	TE2000
Laminin	ThermoFisher Scientific	23017-015
Leibovitz's L-15 Medium with phenol red	Gibco/Fisher Scientific	11-415-064
Leibovitz's L-15 Medium without phenol red	Gibco/Fisher Scientific	21-083-027
Penicillin/streptomycin	ThermoFisher Scientific	15140122
Phenol Red	Sigma Aldich	P0290
Poly-D-Lysine (PDL)	Sigma Aldich	P7280
Steritop 0.22 µm filter	Millipore	S2GPT05RE
Calcium Chloride Dihydrate	Fisher Scientific	C79-500
Glucose	Sigma Aldrich	G7528
HEPES	Sigma Aldrich	H4034