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

To visualize oxidative stress by confocal microscopy, add 1 microliter of N-acetylcysteine stock solution in the corresponding wells of the microslide eight-well chamber plated with organoids.

After a 1-hour incubation, add 1 microliter of Tert-butyl hydroperoxide stock solution in the corresponding wells, and incubate for another 30 minutes. Next, add 1 microliter of the 1.25 millimolar dilution of the fluorogenic probe per well, followed by 1 microliter of 1.25 milligrams per milliliter Hoechst solution.

After another 30-minute incubation, remove the medium without disturbing the BMM, and gently add 250 microliters of warm DMEM without phenol red.

Image the organoids using a confocal microscope, equipped with a thermic chamber and gas supply that detects the fluorogenic probe.

Using the positive control, set up the laser intensity and time exposure for the ROS signal, and check that this signal is lower in the negative control. Next, using an eyepiece, screen the slide to identify the organoids expressing GFP, and adjust the laser intensity.

Set up a z-stack of 25 micrometers, and define positions to obtain a stitched image of the whole organoid to get a section of the organoids showing one layer of cells.