Measurement of reactive oxygen species or ROS is notoriously problematic. In this video, we will demonstrate the reproducible measurement of ROS in response to FC-gamma receptor crosslinking using fluorescent probes and flow cytometry. The main advantages of this technique are the reproducibility of the assay and the use of this method with specific antigenic stimuli, not only mitogens or known ROS inducers.
Dysregulated ROS production is central to the development of many diseases such as chronic granulomatous disease or CGD. Accurately measuring ROS could constitute one part of the molecular diagnosis of CGD. Studying FC-mediated ROS production is important in studying immunodeficiencies, such as CGD, but also in studying auto-immune diseases, or neurodegenerative diseases, where too much ROS leads to oxidative stress and inflammation.
The timing of the assay is critical. I would advise someone trying this protocol for the first time to prepare thoroughly and do a mock experiment if possible. Visual demonstration of this method is critical in order to stress the steps of the assay which need particular attention such as the timing of the assay.
To begin, prepare bone-marrow-derived macrophages using conditioned media as described in the accompanying text protocol. After priming the macrophages overnight, aspirate the supernatant and wash the cells once with PBS. Serum starve the cells by replacing media with the same volume of low-serum DMEM.
For each mouse, one plate will be treated with anti-BSA IgG1 while serum starving while the other will be left untreated. Add only low-serum DMEM to the untreated plates and add low-serum DMEM containing 2.5 micrograms per milliliter of murine anti-BSA IgG1 to the treated plates. Be sure to include one additional untreated plate for each experiment to act as the flow cytometric compensation control.
Incubate the plates four hours at 37 degrees Celsius, 5%CO2. During the four-hour incubation, prepare a 2X solution of the reactive oxygen species probes. For every 10 milliliters of low-serum DMEM needed, add four microliters of an oxidative stress detection reagent and four microliters of a superoxide detection reagent.
Then, prepare the 2X probe solutions containing only the oxidative stress detection reagent or containing only the superoxide detection reagent. After the four-hour incubation, harvest the cells by gently scraping the plates, collecting them in labeled, five-milliliter round-bottomed tubes. Centrifuge the tubes at 750 times g for five minutes.
Be sure to keep track of which cells were treated with murine anti-BSA IgG1. Next, wash the cell pellets with two milliliters of PBS to get rid of any residual anti-BSA from the treated cells. Centrifuge the tubes at 750 times g for five minutes.
Then, aspirate the PBS and resuspend the cell pellet in 600 microliters of low-serum DMEM. From the 600-microliter cell suspension, aliquot 200 microliters into the prelabeled five-milliliter round-bottom tubes. From the untreated cells, take 200 microliters for the tubes labeled unstimulated, 200 microliters for the tubes labeled positive inducer, and 200 microliters for the tubes labeled positive inducer plus inhibitor.
Then, from the anti-BSA IgG1 treated cells, take 200 microliters for tubes labeled FC-gamma receptor crosslinking, and 200 microliters for tubes labeled FC-gamma receptor crosslinking plus inhibitor. From the untreated cells to be used for compensation controls, take 200 microliters for the tube labeled unstained unstimulated, 200 microliters for the green plus inducer control, and 200 microliters for orange plus inducer control. Prepare a 2X positive inducer solution by diluting the pyocyanin one to 100 into each of the 2X probe solutions to a concentration of 500 micromolar.
Then, prepare a 2X BSA solution by diluting the BSA stock solution in the 2X probe solution to obtain a 2X concentration of two micrograms per milliliter. Before starting the specific stimulation, such as FC-gamma receptor crosslinking, make sure that all of the reagents and cells are ready and place the tubes on ice in the order in which they will be stimulated. For flow cytometers with an autosampler, take note of the time that the cytometer takes to analyze one sample and move on to the next.
Be sure to include any mixing and probe-washing steps. Timing is very critical for this assay. In order for every condition to be well controlled, the stimulation needs to be carried out in exactly 30 minutes for each condition.
Stimulate the cells in order and incorporate the lag time between sample acquisitions by the flow cytometer. If performing manual compensation at this point, stimulate control tubes to be used for compensation by adding 200 microliters of the 2X probe solution containing the oxidative stress detection reagent and the inducer into the tubes marked green plus inducer. Then, add 200 microliters of 2X probe solution containing the superoxide detection reagent and the inducer into the tubes marked orange plus inducer.
Incubate the cells for 30 minutes in the dark. Using the flow cytometry software, generate and label three sample files for the control, unstained, untreated, the green plus inducer, and the orange plus inducer samples, making sure to indicate the channels and parameters to be analyzed. Also, enter desired stop conditions.
Once the control samples have been set up, generate and label a similar set of files for the experimental samples. Now, run the unstained, untreated sample. Open a dot plot for forward scatter on the X-axis versus side scatter on the Y-axis and draw a gate around the cells of interest, excluding dead cells and debris.
Next, use this cell's gate to open another dot plot of the first fluorescence marker, FL1, on the X-axis, versus the second fluorescence marker, FL2, on the Y-axis. Draw an initial quadrant gate and then adjust the quadrant gate so that the events appear on the lower left quadrant of the FL1 versus FL2 plot. When finished, run the green plus inducer sample.
Adjust the voltage so that the events appear on the lower left and right quadrants of the FL1 versus FL2 plot. Then, apply this compensation matrix to all three sample files. Now, run the orange plus inducer sample.
Adjust the voltage so that the events appear on the upper and lower left quadrants of the FL1 versus FL2 plot, then apply this compensation matrix to all three sample files. Check each compensation file and ensure that unstained, untreated events appear on the lower left quadrant, the green plus inducer events appear on the lower left and right quadrants, and the orange plus inducer events appear on the upper and lower left quadrants of the FL1 versus FL2 plot. Then, apply the compensation matrix to all of the experimental sample files.
Once manual compensation has been performed and an experimental template has been obtained, move on to the experimental samples. Before treating the cells with a positive inducer or conducting FC-gamma receptor cell stimulation, mark which tubes will get an ROS inhibitor. Treat these cells with the ROS inhibitor at least 30 minutes prior to the positive inducer or FC-gamma receptor stimulation by adding one microliter of the inhibitor to 200 microliters of resuspended cells for a final concentration of five millimolar.
For unstimulated cells, treat the cells with 200 microliters of the 2X probe solution without any stimulus added to the 200 microliters of cell suspension labeled stained, unstimulated. For positive controls, treat the cells with 200 microliters of the 2X positive inducer solution to 200 microliters of cell suspension labeled positive inducer or positive inducer plus inhibitor. Finally, for cells stimulated by FC-gamma receptor crosslinking, treat them with 200 microliters of the 2X BSA solution added to 200 microliters of the cell suspension labeled FC-gamma receptor crosslinking or FC-gamma receptor crosslinking plus inhibitor.
Incubate the cells for 30 minutes in the dark. Following incubation, analyze the samples in the order in which they were stimulated, using the flow cytometer and the analysis templates generated during the initial compensation steps. The data shown here demonstrate the flow cytometric detection of reactive oxygen species production resulting from stimulation of macrophages through the FC-gamma receptor.
There is a marked increase in FL1 and FL2 fluorescence when cells are stimulated with FC-gamma receptor crosslinking agent. When cells were treated with the reactive oxygen species inhibitor prior to FC-gamma receptor crosslinking, this increased fluorescence is brought back to basal levels. In contrast, these dot plots present an unsuccessful experiment, where suboptimal reactive oxygen species production as a result of FC-gamma receptor stimulation was observed.
To highlight the large differences between the expected percentages, or MFI increases, this data shows what samples with a minimal increase in FL1 and FL2 fluorescence looks like. The current protocol utilizes a 24-hour priming step. When comparing a 24-hour versus a 48-hour priming time, there was no marked difference in the percentage of cells positive for the green oxidative stress reagent.
However, increasing the priming time to 48 hours did increase the percentage of cells positive for the orange fluorescence. The most important things to remember when performing this procedure is planning ahead, and being consistent about the timing of the assay to get reproducible results.
