Fluorescent Probe Imaging Assay: A Technique to Visualize Oxidative Stress in the Reactive Oxygen Species Inducer-Treated Cultured Intestinal Organoid Cells

Abstract

Source: Stedman, A. et al., Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe. J. Vis. Exp. (2021).

This video describes the technique of visualizing oxidative stress in cultured organoids. This work helps in understanding the level of oxidative stress due to ROS accumulation in the various cell types present in an organoid after treatment with different ROS modulators.

Protocol

1. Visualization of oxidative stress in 3D organoids by confocal microscopy

1. Take the organoids plated in the µ-Slide 8 well chambers and add 1 µL N-acetyl cysteine or NAC stock solution in the corresponding wells to obtain a final concentration of 1 mM.
2. Incubate for 1 h at 37 °C and 5% CO₂.
3. Add 1 µL tBHP stock solution in the corresponding wells to obtain a final concentration of 200 µM.
4. Incubate for 30 min at 37 °C and 5% CO₂.
5. Add 1 µL per well of the 1.25 mM dilution of the fluorogenic probe to obtain a final concentration of 5 µM.
6. Add 1 µ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Mice
Lgr5-EGFP-IRES-creERT2 (Lgr5- GFP)	The Jackson Laboratory
Growth culture medium
Advanced DMEM F12 (DMEM/F12)	ThermoFisher	12634010
Penicillin-Streptomycin (P/S)	ThermoFisher	15140122	Stock Concentration: 100x (10,000 units/mL of penicillin and 10,000 µg/ mL of streptomycin)
FLuoroBrite DMEM (DMEM no phenol red)	ThermoFisher	A1896701
Hoechst 33342	ThermoFisher	H3570	stock at 10 mg/mL
Matrigel Growth Factor Reduced, Phenol Red Free (Basement Membrane Matrix)	Corning	356231	once received thaw o/n in the fridge, keep for 1h on ice and, make 500 mL aliquots and store at -20 °C
µ-Slide 8 Well chambers	Ibidi	80826
N-acetylcysteine (NAC)	Sigma	A9165
tert-Butyl hydroperoxide (tBCHP)solution (70%wt. In H2O2)	Sigma	458139
Programs and Equipment
Fiji/ImageJ			https://imagej.net/software/fiji/ downloads images generation
Observer.Z1	Zeiss		confocal system
Opterra (swept-field confocal)	Bruker		confocal system
high speed EMCCD Camera Evolve Delta 512	Photometrics		confocal system
Prism	GraphPad Software		statistical analysis