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

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 µL per well of the 1.25 mg/mL dilution of Hoechst to obtain a final concentration of 5 µg/mL.
7. Incubate for 30 min at 37 °C and 5% CO₂.
8. Remove the medium without disturbing the BMM. Gently, add 250µL of warm DMEM without phenol red.
   NOTE: If a long-term acquisition is planned, add growth factors compounds to DMEM without phenol red.
9. Image the organoids using a confocal microscope equipped with a thermic chamber and gas supply that detects the fluorogenic probe (ROS).
   NOTE: The excitation/emission (ex/em) for the fluorogenic probe is 644/665, ex/em for Hoechst (nuclei) is 361/486, and ex/em for GFP (intestinal stem cell from the Lgr5-GFP mice) is 488/510. A 63x oil immersion objective is used to detect signals in stem cells. Do not change laser settings between samples. A 20x objective might be used to allow an overview of ROS production.
10. Use the positive control to set up laser intensity and time exposure for the ROS signal and check that this signal is lower in the negative control.
11. Using the eyepiece, screen the slide to identify the GFP organoids expressing and adjust laser intensity.
    NOTE: This step is manually performed.
12. Define positions to obtain a stitched image of the whole organoid. Set up a z-stack of 25 µm (step size 5 µm) to get a section of the organoids showing one layer of cells.
    NOTE: Refer to the microscope user manual to optimize the setup. Using living cells, the acquisition should be done within 1 h after the end of the incubation.
13. Open the images in an open-source image processing software (see Table of Materials).
14. Go through the z-stack and choose the section in which the middle of the organoids is well represented and create a new image with the selected area.
15. Quantify the images as per the following steps.
    1. Select the freehand line tool.
    2. Draw a line following the nuclei.
       NOTE: Select only regions presenting GFP-positive cells if only stem cells are analyzed.
    3. Increase the line width to cover the cell layer with the line without including the luminal debris.
    4. Select the channel for the ROS signal and measure the fluorescence intensity in the selected region and annotate the values.
    5. Draw a line where there is no signal and measure the fluorescent intensity of the background that will be subtracted from the previous value to get the final intensity.