Flow Cytometric Measurement Of ROS Production In Macrophages In Response To FcγR Cross-linking

Summary

This study demonstrates the use of flow cytometry to detect reactive oxygen species (ROS) production resulting from activation of the FcγR. This method can be used to assess changes in the antimicrobial and redox signaling function of phagocytes in response to immune complexes, opsonized microorganisms, or direct FcγR cross-linking.

Abstract

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in response to various immune stimuli. ROS generated during immune activation exerts potent antimicrobial activity primarily through the ability of ROS to damage DNA and proteins, causing death of microorganisms. Being able to measure ROS production reproducibly and with ease is necessary in order to assess the contribution of various pathways and molecules to this mechanism of host defense. In this paper, we demonstrate the use of fluorescent probes and flow cytometry to detect ROS production. Although widely used, fluorescent measurement of ROS is notoriously problematic, especially with regards to measurement of ROS induced by specific and not mitogenic stimuli. We present a detailed methodology to detect ROS generated as a result of specific FcγR stimulation beginning with macrophage generation, priming, staining, FcγR cross-linking, and ending with flow cytometric analysis.

Introduction

Reactive oxygen species (ROS) are reactive molecules or free radicals that are by-products of aerobic respiration (reviewed in ¹). These include the superoxide anion, peroxide, hydrogen peroxide, hydroxyl radical, and hydroxyl ions, among others. Under normal physiologic conditions, ROS are produced mainly by the mitochondria and nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and are rapidly detoxified by various enzymes and proteins such as superoxide dismutase and glutathione. An exaggerated production of ROS or a defect in the ability to remove ROS can result in oxidative stress, whereby reactive oxygen species promote the damage of proteins, lipids, and DNA leading to cellular stress or death and pathological disease states. However, it is currently appreciated that ROS can also act as signaling molecules (redox signaling), and ROS-mediated modification of various molecules and pathway intermediates can influence cellular metabolism, proliferation, survival, inflammatory signaling, and aging². In phagocytic cells, ROS plays an essential role in providing antimicrobial activity during the so-called “respiratory burst”^1,3,4,5,6. During the response of phagocytes to external stimuli, components of the NADPH oxidase complex (p40^phox, p47^phox, p67^phox) translocate from the cytosol to the phagosomal membrane containing the gp91phox and p22phox subunits, and together with the actions of Rac1/2, form a fully functional NADPH oxidase enzyme complex. The assembled NADPH oxidase then utilizes NADPH to reduce oxygen to superoxide within the phagosomal vacuole. Superoxide anions can directly cause damage or be dismutated into hydrogen peroxide. Both superoxide and hydrogen peroxide can react with other molecules to generate highly reactive hydroxyl radicals. Damage is mediated by reaction of these ROS with iron-sulfur clusters on proteins or by causing base oxidation of DNA, ultimately leading to restricted microbial metabolism or death of the microbe⁵. The importance of the NADPH oxidase enzyme complex and ROS produced during the respiratory burst is illustrated clinically in patients with Chronic Granulomatous Disease (CGD)^7,8,9,10. Individuals with CGD possess mutations in gp91phox, resulting in a lack of ROS production and susceptibility to recurrent infections with bacteria and fungi which are not usually a concern with immunocompetent individuals. Therefore, whether studying oxidative stress, redox signaling, or host defense, being able to measure ROS production in real-time is a useful endeavor.

Multiple assays have been utilized to measure ROS production or the results of oxidative stress^11,12,13. Among these, one of the most widely used is the fluorescent probe 2’,7’ dichlorodihydrofluorescein diacetate (DCFH₂-DA)¹⁴. This molecule is colorless and lipophilic. Diffusion of DCFH₂-DA across the cell membrane allows it to be acted upon by intracellular esterases, which deacetylates it into DCFH_2, rendering it cell impermeable. The actions of multiple types of ROS (hydrogen peroxide, peroxynitrite, hydroxyl radicals, nitric oxide, and peroxy radicals) on DCFH₂ oxidize it into DCF which is fluorescent (reported Ex/Em: 485-500 nm/515-530 nm) and can be detected using a flow cytometer equipped with a standard filter set for fluorescein (FL1 channel). Superoxide does not strongly react with DCFH₂ but can react with another probe dihydroethidium (DHE) to yield the fluorescent product 2-hydroxyethidium (as well as other fluorescent superoxide-independent oxidation products)¹⁵. The fluorescent products of DHE oxidation can be detected using an excitation wavelength of 518 nm and an emission wavelength of 605 nm (FL2 channel). Although relatively simple to use, utilization of these probes for detection of ROS requires knowledge of their limitations and careful incorporation of staining procedures and controls into the specific assay being performed in order to have valid experimental results and conclusions. The following protocol demonstrates the use of a commercially available kit employing these 2 probes designed to measure ROS by flow cytometry. We stain primed bone marrow-derived macrophages with these probes and induce ROS production through FcγR cross-linking. We present representative data obtained using this protocol and stress appropriate precautions that must be undertaken for successful experimentation.

Protocol

The protocol for animal handling was approved by the Institutional Animal Care and Use committee (IACUC) of University of Central Florida.

1. Generation of bone marrow derived macrophages (BMDMs)

1. Culture media preparation
   1. Prepare D10F base media: To Dulbecco’s Modified Eagle Medium (DMEM), add 10% heat inactivated fetal bovine serum (FBS), 1 mM sodium pyruvate, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 0.05 mM β-mercaptoethanol.
      NOTE: Although it is best to use fresh D10F media, D10F base media can be prepared up to two weeks in advance to be used for multiple experiments within a week. For one mouse, around 175 mL of D10F base media is needed (25 mL to flush the bones and 150 mL to make macrophage differentiation media)
   2. Prepare LADMAC growth media: To Eagle's Minimum Essential Medium (EMEM), add 10% heat inactivated FBS and 2 mM L-glutamine. Prepare the media fresh, on the day you plan to start your cultures.
      NOTE: One liter of LADMAC growth media will result in approximately (19) 50 mL aliquots of LADMAC-conditioned media.
   3. Prepare complete DMEM media: To DMEM, add 10% heat inactivated FBS and 1x Antibiotic-Antimycotic. Prepare the media fresh, on the day BMDMs will be harvested.
      NOTE: For one mouse, around 100 mL of DMEM complete media will be required. This includes media used for priming the cells.
2. Preparation of LADMAC conditioned medium
   1. Grow LADMAC cells to confluency in a 10 cm dish using 10 mL of LADMAC growth media (defined in 1.1.2). Incubate cells at 37 °C, 5% CO_2.
   2. At confluency, detach cells by forcefully dispensing media over cells. Passage cells by adding 1 mL of detached cells into T-175 flasks containing 100 mL of LADMAC growth media. Incubate cells until completely confluent at 37 °C, 5% CO₂ (approximately one week). This way, one 10 cm dish can yield ten T-175 flasks or approximately 1 L of conditioned media.
   3. Once the cells are ready, collect the conditioned media and centrifuge at 350 x g for 5 min to pellet unwanted cells. Filter the collected supernatants through a 0.2 μm filter and store 50 mL aliquots at -80 °C. Aliquots of LADMAC-conditioned media can be kept at -80 °C for months with minimal loss of activity.
      NOTE: If large experiments are anticipated, prepare large batches of LADMAC-conditioned media at the same time to ensure consistency. If this is not possible, use aliquots derived from the same batch or lot of LADMAC-conditioned media for each experiment.
3. Preparation of bone marrow derived macrophages
   1. On the day of the experiment, prepare fresh macrophage differentiation media by adding 25% of LADMAC conditioned media into previously prepared D10F media (1 part LADMAC conditioned media into 3 parts D10F). Do not prepare this media in advance! Only prepare as much media as needed for the BMDM culture.
   2. Day 1: Isolate mouse bone marrow cells according to the previously described protocol¹⁶ with a modification to flush the bone marrow from femurs and tibias using D10F base media. Use 2.5 mL of D10F media to flush each end of the (4) bones. Flush bone marrow cells directly into a pre-wet 40 μm cell strainer placed on top of a 50 mL tube. The remaining media (~5 mL) can be used to rinse out the cell strainer.
   3. Centrifuge collected bone marrow cells at 350 x g for 5 min. Discard the supernatant and resuspend the cells in a total of 30 mL of macrophage differentiation media (per mouse).
   4. Prepare and label (6) sterile 10 cm Petri dishes. Plate 5 mL of macrophage differentiation media into each into Petri dish. Aliquot 5 mL of resuspended bone marrow cells in macrophage differentiation media into each Petri dish for a total volume of 10 mL per dish. Incubate for 5 days at 37 °C, 5% CO_2.
   5. Day 5: Aspirate media from the cells. Wash once with 1-2 mL of PBS and add 10 mL of freshly prepared macrophage differentiation media. Incubate for an additional 3 days.
   6. Day 8: Proceed to harvesting BMDMs as described below.

2. Harvesting, seeding and priming of BMDMs

1. After 7 days of growing the BMDMs in the macrophage differentiation media, remove the supernatant. Wash adherent macrophages once with 1-2 mL of PBS. Aspirate the PBS.
2. Dispense 1 mL of 0.25% Trypsin-EDTA into each macrophage plate and leave them in the 37 °C incubator for 5-10 min with frequent tapping to detach the cells. Dissociate trypsinized macrophages by pipetting up and down using a P1000 pipette and collect macrophages in 50 mL tubes containing 10-15 mL of complete DMEM media. Wash the plate with additional 2 mL complete DMEM media to harvest any remaining macrophages.
   CAUTION: Do not leave macrophages in trypsin for more than 10 min.
3. Centrifuge the 50 mL tubes at 350 x g for 5 min. Discard the supernatant. Gently dissociate the pellet and resuspend the cells in 10 mL of complete DMEM media. Count macrophages using a hemocytometer in the presence of a viability dye such as trypan blue as follows:
   1. For example, mix 90 µL of Trypan blue with 10 µL of cells for a 1:10 dilution.
   2. Of this mixture, load 10 µL into a hemocytometer and count the central square (5 x 5 grid). The total cell count = count x 10⁴ x dilution factor (10) x volume (10 mL).
4. Adjust the cell suspension to be 1 million/mL in complete DMEM media and plate 4 mL of this cell suspension into each 6 cm dish.
   NOTE: A minimum of 3 plates is required per experiment. One plate of cells will be left unstimulated, a second set of cells will be stimulated through cross-linking of FcγRs, and the third plate of cells will be used for all of the additional experimental and flow cytometric controls.
5. Incubate the plates overnight at 37 °C, 5% CO₂.
6. The following day, aspirate the supernatant, wash cells once with 1-2 mL of PBS. Add 4 mL of complete media containing 100 ng/mL mouse IFN-γ to each plate. Incubate the plates overnight at 37 °C, 5% CO₂.
7. If desired, take an aliquot of the cells to assess appropriate generation of BMDMs by flow cytometry. Use 1x10⁶ cells per test and prepare polystyrene FACs tubes for the following conditions: isotype control, stained with F4/80 (or alternatively, stained with CD11b).
8. Prepare flow cytometry staining buffer by adding 0.1% FBS into 1x PBS. Use only this amount of FBS so as not to negate the effects of serum-starvation in later steps.
9. Aliquot 1 x 10⁶ cells per tube and centrifuge at 750 x g for 5 min.
10. Wash once by decanting or aspirating supernatant and resuspending cells in 2 mL of flow cytometry buffer. Centrifuge at 750 x g for 5 min.
11. Aspirate supernatant and resuspend cells in 100 µL of flow cytometry staining buffer. Add 1 µL of anti-mouse CD16/32 Fc blocking antibody to each tube. Incubate on ice for 5 min.
12. Without washing, add 2 µL of FITC anti-mouse F4/80 or 1 µL of APC anti-mouse CD11b antibody to the “stained” tubes and a similar amount of the corresponding isotype control antibody to “isotype” tubes. Incubate on ice, in the dark, for 30 min.
13. Wash cells by adding 2 mL of PBS directly to the cells. Centrifuge at 750 x g for 5 min.
14. Aspirate supernatant and resuspend cells in 150 µL of PBS.
15. Acquire samples on a flow cytometer using a stop condition of 100 µL or 10,000 events on the gate of interest and ensure that FSC, SSC, FL1 (FITC anti-mouse F4/80) or FL4 (APC anti-mouse CD11b) channels are selected for the parameters to be analyzed.
16. Using the flow cytometry software, open a dot plot for FSC (on the x-axis) vs SSC (on the y-axis) and draw a gate around the cells of interest, excluding dead cells and debris (dead cells and debris are much smaller events than the main cell population and appear on the lower left of the plot).
17. Gating on the cells of interest, open a histogram plot with FL1 (FITC anti-mouse F4/80) or FL4 (APC anti-mouse CD11b) on the x-axis.
18. Run the “isotype” sample. Generate a marker gate so that majority of “isotype” sample events are to the left of the gate (<1% positive). Apply this template to the stained sample file.
19. Run the “stained” sample. Successful generation of BMDMs will result in >95% expression of FITC anti-mouse F4/80 or APC anti-mouse CD11b (Figure 1A), while incorrect culture conditions may result in sub-optimal generation of macrophages (Figure 1B).
    NOTE: If generating BMDMs from multiple genotypes, take note of the expression of these markers for each batch of BMDM generated, in the case that this may be grounds for excluding a sample from analysis when ROS generation in response to stimulus is assessed.

3. Reagent and material preparation for ROS measurement

1. Reconstitute the lyophilized oxidative stress detection reagent in 60 µL of anhydrous DMF to yield a 5 mM stock solution. Gently mix before use.
   NOTE: It is stated in the ROS-ID kit manual that the shelf life of the reconstituted reagent is about 1 week at -20 °C. Aliquoting the reagent immediately after reconstitution into small light-proof vials minimizes its oxidation and maximizes the shelf life at -20 °C.
2. Reconstitute the lyophilized superoxide detection reagent in 60 µL of anhydrous DMF to yield a 5 mM stock solution. Gently mix before use.
   CAUTION: As stated in the kit manual, treat both detection reagents as possible mutagens, handle each with care, and dispose of properly.
3. Reconstitute the ROS inducer (Pyocyanin) in 20 µL of anhydrous DMF to yield a 50 mM stock solution.
4. Reconstitute the ROS Inhibitor (N-acetyl-L-cysteine) in 123 µL of deionized water to yield a 0.5 M stock concentration.
5. Label 5 mL round polystyrene tubes (flow cytometry tubes) with the experimental controls and conditions. (See 4. Assay conditions and controls)
6. Prepare low serum DMEM (no phenol red) by adding 0.1% FBS to phenol red-free DMEM.
7. Aliquot the anti-BSA antibody into small aliquots (depending on the usage) and store them at -80 °C.
8. Prepare stock solution of 100 mg/mL BSA in HBSS (Hanks’ balanced salt solution) or PBS. Aliquot into 100 µL aliquots and store at -20 °C.
9. Prepare a 2x solution of the ROS probes (2x probe solution): for each 10 mL of low serum DMEM, add 4 µL of oxidative stress detection reagent (green dye) and 4 µL of superoxide detection reagent (orange dye).
10. Prepare 2x probe solutions containing only oxidative stress detection reagent (2x probe solution, green only), or containing only superoxide detection reagent (2x probe solution, orange only). Prepare only the amount of reagent needed for the experiment and always prepare the 2x solution immediately prior to use.
    NOTE: To prepare smaller volumes of 2x detection solution, use an intermediate 1:10 dilution of both Green and Orange detection reagents prior to final dilution in DMEM. For example, to prepare 1 mL of a 2x detection solution, dilute 1 µL of each probe into 9 µL of DMEM (1:10 intermediate dilution). Dilute 4 µL of this 1:10 intermediate dilution into 1 mL of DMEM.

4. Assay conditions and controls

1. Include the following experimental controls for flow cytometric compensation for each experiment:
   a) Unstained and unstimulated cells.
   b) Cells stained only with the oxidative stress detection reagent (green reagent) and treated with ROS inducer.
   c) Cells stained only with the superoxide detection reagent (orange reagent) and treated with ROS inducer.
2. For each mouse or biological replicate, include the following 6 conditions:
   a) Unstained and unstimulated cells
   b) Stained and unstimulated cells
   c) Stained cells treated with positive inducer
   d) Stained cells treated with positive inducer and ROS inhibitor
   e) Stained cells activated through FcγR cross-linking
   f) Stained cells activated through FcγR cross-linking and treated with ROS inhibitor
3. Calculate the amount of 2x probe solution needed based on the number of mice and conditions (200 µL of the 2x probe solution will be used for each condition requiring staining).
   NOTE: For an assay using 6 mice, 5 conditions requiring staining with the probes will be needed per mouse. This brings the total number of conditions to 30. Thus, the total amount of 2x probe solution needed is at least 6 mL (30*200 µL).

5. Cell preparation

1. After priming the macrophages overnight, aspirate the supernatant. Wash the cells once with PBS.
2. 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 untreated plates. Add low serum DMEM containing 2.5 µg/mL of murine anti-BSA IgG₁ to treated plates.
   NOTE: One additional untreated plate is needed for each experiment for the flow cytometric compensation controls.
3. Incubate the plates for 4 h at 37 °C, 5% CO₂.
4. After serum starving, harvest the cells by gentle scraping or by using 0.2 mM EDTA in PBS. Collect them in labeled 5 mL round bottom tubes and centrifuge them at 750 x g for 5 min. Keep track of which cells were treated with murine anti-BSA IgG₁.
5. Wash cell pellets once with 2 mL of PBS to get rid of any residual anti-BSA from the treated cells.
6. Resuspend cell pellet in 600 µL of low serum DMEM.
7. From the 600 µL cell suspension, aliquot 200 µL into the pre-labelled 5 mL round bottom tubes as follows:
   1. From the untreated cells, take 200 µL for tubes labelled “unstimulated”, 200 µL for tubes labelled “positive inducer” and 200 µL for tubes labelled “positive inducer + inhibitor”
   2. From the anti-BSA IgG₁ treated cells, take 200 µL for tubes labelled “FcγR crosslinking/FcγR XL” and 200 µL for tubes labelled “FcγR XL + inhibitor”
   3. From the untreated cells to be used for compensation controls, take 200 µL for the tube labelled “unstained unstimulated”, 200 µL for the “green + inducer” control and 200 µL for “orange + inducer” control.
      NOTE: If the cells from 5.7.1 and 5.7.3 are derived from the same mouse, the same “unstained unstimulated” cells can be used for the experimental and compensation control. If not, an additional 200 µL of cells will be needed for an “unstained unstimulated” experimental control for a).
8. Keep the tubes on ice until staining and stimulation. During serum starvation and binding of IgG_1, prepare the probes, specific stimuli, and inducers as outlined below.
   NOTE: If performing the assay for the first time, if no template is available, or no after-the-fact compensation is available on the flow cytometer and corresponding software, stimulate and stain compensation controls and run these on the flow cytometer to perform manual compensation prior to addition of stimuli to experimental samples.

6. Performing the assay

1. Prepare 2x positive inducer solution by diluting the Pyocyanin 1:100 in the 2x probe solution (prepared in step 3.9) to obtain a 2x concentration of 500 µM of pyocyanin (final concentration will be 250 µM). Also, dilute pyocyanin 1:100 into each of the “2x probe solution, green only” and “2x probe solution, orange only” tubes prepared in 3.10.
   NOTE: Both conditions labelled with “positive inducer” and “positive inducer + inhibitor” will be treated with the positive inducer. Plan accordingly when calculating the amount of 2x positive inducer solution to prepare. For example: If performing the assay with 3 mice, 6 conditions (3*2) will be treated with positive inducer, so prepare 6*200 µL =1.2 mL of 2x positive inducer solution.
2. Prepare a 2x BSA solution by diluting the BSA stock solution in 2x probe solution to obtain a concentration of 2 µg/mL (final concentration will be 1 µg/mL).
   NOTE: Both conditions labelled with “FcγR XL” and “FcγR XL + inhibitor” will be treated with the 2x BSA solution. Plan accordingly when calculating the amount of solution to prepare. For example: If performing the assay using 3 mice, 6 (3*2) conditions will be treated with BSA solution and so 6*200 µL or 1.2 mL of 2x BSA solution will be needed.
3. Before starting the specific stimulation (FcγR crosslinking), make sure that all the reagents and cells are ready. Place the tubes on ice in the order they will be stimulated.
4. 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, including any mixing and probe washing steps (for example 3.5 min).
   NOTE: Timing is very critical for this assay. In order for every condition to be well controlled, the stimulation needs to be carried out for the exact time (30 min) for each condition. Stimulate cells in order and incorporate the lag time between sample acquisition by the flow cytometer. For example, if the time needed for the cytometer to analyze one sample and move on to the next is 3.5 min, stimulate cells in the order they will be analyzed every 3.5 min.
5. If performing manual compensation at this point, stimulate control tubes to be used for compensation (step 5.7.3). Add 200 µL of “2x probe solution, green only” containing inducer (from 6.1) into the tubes marked “green+inducer” (step 5.7.3). Add 200 µL of “2x probe solution, orange only” containing inducer (step 6.1) into the tubes marked “orange+inducer” (step 5.7.3).
6. Incubate the cells for 30 min at 37 °C, 5% CO₂ in dark.
7. Using the flow cytometry software, generate and label 3 sample files for the control “unstained, untreated”, “green+inducer”, and “orange+inducer” samples, making sure to indicate the channels/parameters to be analyzed (FSC, SSC, FL1, FL2) and desired stop conditions (100 µL, 3 min, etc.). Generate and label a similar set of files for experimental samples.
8. Run the “unstained, untreated” sample. Open a dot plot for FSC (on the x-axis) vs SSC (on the y-axis) and draw a gate around the cells of interest, excluding dead cells and debris (dead cells and debris are much smaller events than the main cell population and appear on the lower left of the plot).
9. Using this “cells” gate, open another dot plot of FL1 (x-axis) vs FL2 (y-axis). Draw an initial quadrant gate. Adjust the quadrant gates so that the events appear on the lower left quadrant of the FL1 vs FL2 plot.
10. Run the “green+inducer” sample. Adjust the voltage so that the events appear on the lower left and right quadrants of the FL1 vs FL2 plot. Apply this compensation matrix to all 3 sample files.
11. Run the “orange+inducer” sample. Adjust the voltage so that the events appear on the upper and lower left quadrants of the FL1 vs FL2 plot. Apply this compensation matrix to all 3 sample files.
12. Check each compensation file and ensure that “unstained, untreated” events appear on the lower left quadrant, “green+inducer” events appear on the lower left and right quadrants, and “orange+inducer” events appear on the upper and lower left quadrants of the FL1 vs FL2 plot. Apply the compensation matrix to all of the experimental sample files.
    1. Ensure that appropriate compensation is applied to all the control sample files before applying the compensation matrix to all of the experimental sample files. Refer to Figure 2 for representative data showing uncompensated and correctly compensated samples.
       NOTE: Many cytometers designate FL1 as the standard FITC/GFP channel (excited by the blue 488 nm laser, and detected with a 530/30 filter set) and FL2 as the standard PE channel (excited by the blue 488 nm laser, and detected with a 585/40 filter set). We use this same convention but caution new flow cytometry users to consult with their flow cytometry core manager to ensure that their cytometer is similarly configured and that the appropriate channels are used to detect these probes. Depending on the model of flow cytometer and accompanying software, it may be possible to perform compensation before or after samples have been acquired. If after-the-fact compensation is possible, the compensation step can be performed after all experimental samples have been acquired by the cytometer. For cytometers with a dynamic range, where PMT voltage adjustments are not needed, a compensation experiment can also be performed on a separate day and the experimental template (including the compensation matrix) can be saved to reduce the time needed to perform manual compensation.
13. Once manual compensation has been performed and an experimental template is obtained, start treatment of experimental samples. Before treating with positive inducer or FcγR cell stimulation, mark which tubes get ROS inhibitor. Treat these cells with the ROS inhibitor at least 30 min prior to positive inducer or FcγR stimulation.
14. To add the ROS inhibitor, prepare a final concentration of 5 mM by adding 1 µL of the inhibitor to 200 µL of resuspended cells (without anti-BSA IgG₁).
15. Treat the cells with the stimulus (or positive inducer) and load the cells with the ROS probes as follows:
    1. For unstimulated cells: add 200 µL of 2x probe solution without any stimulus to the 200 µL of cell suspension labelled “stained, unstimulated”.
    2. For positive controls: add 200 µL of 2x positive inducer solution (prepared in step 6.1) to 200 µL of cell suspension labelled “positive inducer” or “positive inducer + inhibitor”.
    3. For cells stimulated by FcγR cross-linking (specific stimulus): add 200 µL of 2x BSA solution (prepared in 6.2) to 200 µL of cell suspension labelled “Fcγr XL” or “FcγR XL + inhibitor”.
       NOTE: Remember to incorporate the lag time between sample analysis by adding stimulus every x min where x is the lag time between acquisition of one sample and the next.
16. Incubate the cells for 30 min at 37 °C, 5% CO₂ in dark. Analyze the samples in the order they were stimulated using a flow cytometer equipped with an autosampler. Use the analysis templates generated during the initial compensation steps. Do not wash cells prior to analysis.

7. Flow cytometry data analysis and anticipated results

1. Within the flow cytometry software, open the previously generated analysis files for the experimental samples (templates were generated in steps 6.7-6.12 and samples were run in step 6.16). Ensure that the compensation matrix from performing manual compensation was correctly applied to the experimental samples.
   NOTE: For flow cytometers and flow cytometry software capable of after-the-fact compensation, if the compensation matrix was not previously applied to the experimental sample files, apply it at this point.
2. Similar to verifying correct manual compensation, ensure that the controls within the experimental samples behave as expected.
   1. Ensure that the “unstained, untreated” events will appear on the lower left quadrant of the FL1 vs FL2 plot, that the “positive inducer” events show increased fluorescence in the upper left, upper right, and lower right quadrants of the FL1 vs FL2 plot, and that “positive inducer + ROS inhibitor” events show a reduction in fluorescence in upper left, upper right, and lower right quadrants of the FL1 vs FL2 plot compared to the fluorescence observed with the “positive inducer” sample.
   2. If controls within the experimental samples do not show any increased fluorescence, check that all assay steps were performed, verify appropriate generation and priming of bone marrow-derived macrophages (2.7-2.19), and repeat the experiment. If controls within the experimental samples show the expected trends, proceed to analyze experimental samples stimulated through the FcγR (Figure 3A).
3. Verify that the FcγR-specific stimulus is working appropriately. Run the following samples from a C57BL/6J WT mouse: “unstained, unstimulated”, “stained, unstimulated”, “stained, stimulated through the FcγR”, and “stained, stimulated through the FcγR + ROS inhibitor”. These correspond to samples 4.2a, 4.2b, 4.2e, and 4.2f in section 4, respectively.
   1. Ensure that the “unstained, unstimulated” events will appear on the lower left quadrant of the FL1 vs FL2 plot, that “stained, unstimulated” events will also appear on the lower left quadrant of the FL1 vs FL2 plot, that “stained, stimulated through the FcγR” events show increased fluorescence in the upper left, upper right, and lower right quadrants of the FL1 vs FL2 plot, and that “stained, stimulated through the FcγR + ROS inhibitor” events will show decreased fluorescence in the upper left, upper right, and lower right quadrants of the FL1 vs FL2 plot compared to the “stained, stimulated through the FcγR” sample (Figure 3A).
   2. If these expectations are not met, for example, the “stained, stimulated through the FcγR” sample shows minimal or no increased fluorescence when compared to the “stained, unstimulated” sample, or the “stained, unstimulated” sample already shows markedly increased levels of fluorescence compared to the “unstained, unstimulated” sample, check that all assay steps were performed properly, verify appropriate generation, priming, and handling of BMDMs (2.7-2.19), and repeat the experiment. If these expectations are met, proceed to the analysis of the rest of the experimental samples.
      NOTE: Cells producing ROS which react with the green probe (hydrogen peroxide, peroxynitrite, hydroxyl radicals, nitric oxide, peroxy radicals, etc.) will appear in the upper right and lower right quadrants of a log FL1 (X-axis) versus a log FL2 (Y-axis) dot plot. Cells producing ROS which react with the orange probe (largely but not exclusively superoxide) will appear in the two upper quadrants of a log FL1 (X-axis) versus a log FL2 (Y-axis) dot plot.
4. Generate individual histograms, gated on the cells of interest, for analysis of FL1 and FL2 fluorescence. Using the “unstained, unstimulated” samples, generate a histogram marker such that all “unstained, unstimulated” events appear to the left of this marker. Apply this plot with the marker to the rest of the experimental samples (Figure 3B).
5. Present the results of the experiment as the percentage of the cells positive for each of the ROS probes or by showing the mean fluorescence intensity (MFI) of the stimulated samples versus control (Figure 3C).

8. Cell surface staining in combination with flow cytometric analysis of ROS production (Optional)

NOTE: This step provides a protocol for staining macrophages with a cell-surface marker prior to stimulation of the FcγR and ROS measurement. This may be useful in assessing ROS production in mixed cell populations. It is important to choose an antibody for macrophage surface marker conjugated to an appropriate fluor that does not interfere with the fluorescence from the oxidative stress or superoxide detection reagents. In this protocol, an antibody for mouse F4/80 conjugated to Alexa fluor 647 is used.

1. Generate BMDMs, harvest and prime them as described previously (sections 1 and 2).
   NOTE: A minimum of three 6 cm plates are needed in order to perform all the experimental controls and conditions as described below. One plate will be treated with anti-BSA IgG₁ while serum starving while the other two plates will be left untreated.
   1. Include 4 experimental controls which will be used for flow cytometric compensation (per experiment):
      a) Unstained and unstimulated cells.
      b) Cells treated with the oxidative stress detection reagent (green reagent) and ROS inducer.
      c) Cells treated with the superoxide detection reagent (orange reagent) and ROS inducer.
      d) Cells stained only with anti-mouse F4/80 conjugated to Alexa 647.
   2. For each mouse or biological replicate, include the following 3 conditions:
      a) stained with F4/80 and left unstimulated
      b) stained with F4/80 and activated through the FcγR
      c) stained with F4/80 and activated through the FcγR and treated with ROS inhibitor
2. After priming the macrophages overnight, aspirate the supernatant. Wash the cells once with 1-2 mL of PBS. Serum starve the cells by replacing the 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 two plates will be left untreated. Incubate the plates for 4 h at 37 °C, 5% CO₂.
3. After serum starving, harvest the cells by gentle scraping or by using 0.2 mM EDTA in PBS. Collect each plate in labeled 5 mL round bottom tubes and centrifuge them at 750 x g for 5 min. Keep track of which cells were treated with murine anti-BSA IgG₁.
4. Wash cell pellets once with 1-2 mL of PBS to get rid of any residual anti-BSA from the treated cells.
5. Resuspend one of the cell pellets from the plate which did not get anti-BSA IgG₁ in 600 µL of low serum DMEM. These cells will not be subjected to cell surface staining. Use these for compensation controls. Aliquot 200 µL of this cell suspension into (3) 5 mL round bottom tubes labeled a) unstained, unstimulated b) green oxidative stress reagent + ROS inducer, c) orange superoxide detection reagent + ROS inducer.
6. Resuspend one of the cell pellets from the plate which did not get anti-BSA IgG_1, in 300 µL of flow cytometry staining buffer (PBS + 0.1% FBS). Aliquot 100 µL of this cell suspension into (2) 5 mL round bottom tubes for staining with Alexa 647 anti-mouse F4/80.
7. Resuspend the cell pellets from the plate which received anti-BSA IgG₁ in 300 µL of flow cytometry staining buffer. Aliquot 100 µL of this cell suspension into (2) 5 mL round bottom tubes for staining with Alexa 647 anti-mouse F4/80.
8. Stain the cells, which were aliquoted in 8.6 and 8.7, with 5 µL of Alexa 647 anti-mouse F4/80 for 30 min on ice, in the dark.
9. After staining, wash cells by adding 2 mL of PBS and centrifuge at 750 x g, aspirate the supernatant, and resuspend each tube in 200 µL of low serum DMEM without phenol red. Set aside one untreated tube to serve as singly stained FL4 control. Prepare probes, inducer, FcγR stimulus, and ROS inhibitors as indicated in sections 3.9, 3.10, 6.1, 6.2, and 6.14.
   1. For cells not receiving anti-BSA IgG₁, label tubes with the following: a) no stimulus or b) ROS inducer.
   2. For cells which received anti-BSA IgG₁, label with the following: a) Fc XL or b) Fc XL + inhibitor.
10. Stimulate the samples to be used for compensation according to their respective treatments as indicated in 6.5-6.6, in the order that they will be read on the flow cytometer, incorporating the lag time in between acquisition of one sample and the next.
11. Incubate the cells for 30 min at 37 °C, 5% CO₂ in dark. Analyze the samples in the order they were stimulated using a flow cytometer equipped with an autosampler.
12. Perform manual compensation as described in 6.7-6.12 and data analysis as described in section 7.
13. Using the flow cytometry software, generate and label 4 sample files for the control “unstained, untreated”, “green + inducer”, “orange + inducer” and “F4/80 stained” samples, making sure to indicate the channels/parameters to be analyzed (FSC, SSC, FL1, FL2, FL4) and desired stop conditions (100 µL, 3 min, etc.).
14. Run the samples and generate dot plots to perform manual compensation.
    1. For cell surface staining, generate two additional plots: FL1 (x-axis) vs FL4 (y-axis) and FL2 (x-axis) vs FL4 (y-axis). Adjust the voltages to ensure that appropriate compensation is applied as shown in Figure 7A. Once all compensation has been properly performed, apply the compensation matrix to all of the experimental sample files.
15. Once manual compensation has been performed and an experimental template is obtained, start treatment of experimental samples as indicated in 6.13-6.15.3 and acquire samples on a flow cytometer as described in 6.16.

Results

Using the protocol outlined within, we present representative data demonstrating flow cytometric detection of ROS production resulting from stimulation of WT C57BL/6J BMDMs through the FcγR. As expected, we observe minimal changes in FL1 or FL2 fluorescence above background levels in unstimulated cells (Figure 3A, compare “stained, unstimulated” vs “unstained, unstimulated” dot plots). We observe a marked increase in FL1 and FL2 fluorescence when cells are stimulated with FcγR cross-linking ...

Discussion

DCFH₂-DA and DHE-based detection of ROS is a widely-used technique^14,15. Ease of use and the adaptability of these ROS probes for kinetic microplate formats, fluorescence microscopy or flow cytometric analysis has contributed to their popularity. However, in our studies of FcγR-mediated macrophage functions, there did not seem to be a standard protocol for performing this assay for flow cytometric analysis of FcγR cross-linked cells. Given th...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-BSA IgG1	Innovative Research	IBSA9E2C2
Alexa Fluor 647 Rat IgG2b, κ Isotype Ctrl Antibody	BioLegend	400626
Anti-mouse CD16/32	BioLegend	101302
Anti-mouse F4/80 antibody conjugated to Alexa Fluor 647	BD Biosciences	565853
Anti-mouse F4/80 antibody conjugated to FITC	BioLegend	123108
Anti-mouse/human CD11b antibodyconjugated to Alexa Fluor 647	BioLegend	101218
beta-mercaptoethanol (BME)	Sigma	M3148-100ml
Bovine Serum Albumin (BSA) FractionV	Fisher	BP1600-100
C57BL/6J	Jackson labs	Stock No.000664
CM-H2DCFDA	Molecular Probes	C6827	Can be a substitute for oxidative stress detection reagent in the Enzo kit
Dihydroethidium (DHE)	Molecular Probes	D11347	Can be a substitute for superoxide detection reagent in the Enzo kit
DMEM 1x	Corning	10-013-CV
DMEM no phenol red	Gibco	31053-028
DMF Anhydrous	Acros Organics	61094-1000
Fetal Bovine Serum (FBS)	VWR	97068-085
FITC Rat IgG2a, κ Isotype Ctrl Antibody	BioLegend	400506
HEPES (1M)	Gibco	15630-080
L glutamine	Gibco	25030-081
LADMAC cells	ATCC	CRL-2420
MEM	Corning	10-010-CV
mouse IFN-g	GoldBio	1360-06-100
N-Acetyl-L-cysteine	EMD Milipore	106425	Can be a substitute for ROS inhibitor/scavenger in the Enzo kit
Novocyte flow cytometer with autosampler	Acea	2060R
Pyocyanin (ROS inducer)	Cayman chemical	10009594	Can be a substitute for inducer in the Enzo kit
ROS-ID total ROS/superoxide detection kit	ENZO	ENZ-51010
Sodium pyruvate (100mM)	Gibco	11360-070
Trypsin-EDTA (0.25%)	Gibco	25200-056