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

Published: April 30, 2023

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 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. Incubate for 1 h at 37 °C and 5% CO2. Add 1 µL tBHP stock solution in the corresponding wells to obtain a final concentration of 200 µM. Incubate for 30 min at 37 °C and 5% CO2.<…

Offenlegungen

The authors have nothing to disclose.

Materials

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

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Fluorescent Probe Imaging Assay: A Technique to Visualize Oxidative Stress in the Reactive Oxygen Species Inducer-Treated Cultured Intestinal Organoid Cells. J. Vis. Exp. (Pending Publication), e21220, doi: (2023).

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