This study investigates the visualization of oxidative stress in intestinal organoids, which mimic gut architecture. By using reactive oxygen species (ROS) inducers and fluorescent probes, researchers can assess oxidative stress levels in different cell types within the organoids.
Intestinal organoids recapitulate the gut-like crypt-villus organization having a central lumen comprising dead cells and debris. The lumen is lined by villus-like cells and budding, crypt-like protrusions harboring stem cells.
Different organoid cells produce varying levels of reactive oxygen species, ROS - by-products of metabolic activities, the accumulation of which leads to oxidative stress.
To visualize oxidative stress in varied cells, begin by taking a multi-well plate containing cultured 3D organoids in a basement matrix. The organoid stem cells express a green fluorescence protein or GFP-tagged protein that helps distinguish them during imaging.
Supplement the wells with tert-butyl hydroperoxide - an ROS inducer - and incubate. The inducer activates the membrane-bound NADPH oxidase and stimulates ROS production inside the cells. Stem cells, being metabolically active, produce more ROS than differentiated cells.
Add ROS-sensitive fluorogenic probes. These lipophilic probes rapidly enter the cells and react with ROS to produce red fluorescence. Treat the organoids with Hoechst dye that selectively stains live cell nuclei. Image the organoids using a confocal microscope.
Organoid cells in the periphery with stained nuclei show varying degrees of red fluorescence, which is high in the GFP-expressing stem cells and low in the differentiated cells. The dead cells with inherently high ROS levels exhibit red fluorescence in the lumen.
The intensity of red fluorescence correlates with variable levels of oxidative stress in different cell types.
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.
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