This study investigates the production of reactive oxygen species (ROS) in mouse intestinal 3D organoids. It explores the effects of ROS modulators on cellular homeostasis and cell viability.
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.
Add 1 microliter of the N-acetyl cysteine stock solution in the negative control wells of the 96-well round-bottom plate containing the organoids.
After a 1-hour incubation, add 1 microliter Tert-butyl hydroperoxide stock solution in the corresponding wells, and incubate for 30 minutes. Then, using a multi-channel pipette, remove the medium without disturbing the attached BMM, and transfer it to another 96-well round-bottom plate. Keep this plate aside.
Next, add 100 microliters of trypsin, and using a multi-channel pipette, pipette up and down five times to destroy the BMM. After a short 5-minute incubation, dissociate the organoids by pipetting up and down a second time.
Spin the plate, and discard the supernatant by inverting the plate. Add the medium previously collected in another 96-well plate, back into the corresponding wells, and re-suspend the cells by pipetting up and down five times.
Next, add 1 microliter of the fluorogenic probe, and incubate for 30 minutes. Then, spin the plate again, and re-suspend the cells with 250 microliters of DAPI solution. Transfer the samples in Flow cytometry tubes, and keep the tubes on ice.
Optimize the forward and side scatter voltage settings on unstained control and laser voltages, for each fluorophore using mono-stained samples. Then, using an appropriate gating strategy, collect a minimum of 20,000 events.
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