Flow Cytometry Assay to Quantify Oxidative Stress: An Assay to Quantify Reactive Oxygen Species in Intestinal Organoids Using Fluorogenic Probes

Intestinal cells produce reactive oxygen species – ROS. Moderate ROS levels maintain cellular homeostasis, while high ROS levels can cause cell death.

To quantify ROS levels in vitro, begin with a culture of mouse intestinal 3D organoids. These organoids exhibit gut-epithelium architecture having crypt-like protrusions harboring green fluorescence-expressing stem cells. They also contain villus-like differentiated cells at the periphery, surrounding the central lumen comprising dead cells and debris. All these cells produce different levels of ROS.

Now, treat the organoids in the designated wells with ROS modulators. Based on their nature, modulators can act as ROS inducers or inhibitors. After incubating for the required duration, remove the spent medium.

Add a proteolytic enzyme to the culture, disrupting the organoids' 3D architecture and releasing different cells in suspension. Next, add a ROS-sensitive fluorogenic probe and incubate.

Upon entering the cells, the probes react with ROS and get oxidized to produce red fluorescence. Treat the cells with DAPI – a DNA-binding fluorescent dye – which selectively stains the nuclei of dead cells – creating a contrast with the live cells.

Using a flow cytometer, analyze the DAPI-negative live cells to obtain a red fluorescence signal corresponding to their ROS level. Depending on the nature of modulators, inducers increase ROS levels while the inhibitors decrease them.