Quantification of Reactive Oxygen Species Using 2′,7′-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in Müller Glial Cells

There are several methods for measure ROS production, or oxidative stress in cells.Among these, 2'7'dichlorofluorescein diacetate probe is one of the most widely used technique for directly measure of redox state.This probe is lipophilic and non-fluorescent.Diffusion of this probe across the cell membrane allow the intracellular esterases cleavage at the two ester bonds, producing a relatively polar and cell membrane-impermeable product.These non-fluorescent molecules accumulate intracellularly, and subsequent oxidation by ROS yields the highly fluorescent product dichlorofluorescein.The oxidation of the probe is the product of action of multiple type of ROS.Which can be detected by flow cytometry or confocal microscopy.In this article, we have used a dichlorofluorescein diacetate probe to measure and quantify ROS by flow cytometry.Transfer 6-well plates into a laminar flow hood from the incubator.Aspirate the supernatant.And wash the cells once with PBS.Add 2 mL of low serum DMEM per well.And incubate the 6-well plate for two hours in the incubator.Treat the cells with the antioxidant for six hours.Treat the cells with the ROS inducer, A or B, for 30 minutes.Aspirate the supernatant.And wash the cell three times with PBS to remove all the stimuli.Turn off the light of the laminar flow hood and work in darkness.Add 1 mL of dichlorofluorescein diacetate probe in DMEM without phenol red.Add 1 mL of DMEM without phenol red to the control autofluorescence control cells.Incubate the 6-well plate for 30 minutes in the incubator.Transfer the plates on ice to the laboratory.Wash the cells three times with 2 mL of cold PBS per well.Add 0.5 mL of cold detaching buffer to each well.Harvest the cells by gently pipetting up and down using a P1000 pipette.Collect them in labeled 1.5 mL tube.And centrifuge them at 600 x g for five minutes at 4 C.Discard the supernatant carefully, and gently dissociate the pellet with tapping.Add 1 mL of FACS buffer to wash the cells to each labeled tube.If it's necessary, use vortex in the lowest intensity for the complete dissociation of the pellet.Centrifuge them at 600 x g for five minutes at 4 C.Repeat this step two times more.Three washes in total.Resuspend cell pellets in 200 L of FACS buffer.Gently dissociate completely the pellet in each tube by using vortex.And transfer to labeled 5 mL for flow cytometer tubes.Keep the tubes on ice until analyze on flow cytometer.Using the flow cytometry software, create a new experiment.Click the specimen and will open a list of tubes.Double-click and rename them.Place the autofluorescence control tube on the aspirator arm, moving the base carefully only to the left.Click Acquire Data and then Record Data, and select the desired stop condition.Draw a gate around the cells of interest, excluding dead cells and debris.Remove the tube.Click Next Tube and run the basal control sample.Click Acquire Data and then in Record Data.Open the software.Add the samples using the Add Samples action button.Click Add Sample.Select the Experimental Date folder and click Choose.Double-click the first sample in your workspace, autofluorescence control in this case.Draw a polygon gate to isolate the cell of interest, excluding dead cells and debris.Change plot to a histogram.Double-click within the M