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

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.