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Annexin V and Sytox Green are widely used markers to evaluate apoptosis in various cell types using flow cytometry and fluorescent microscopy. Recently, a novel fluoroprobe MitoSOX Red was introduced for selective detection of superoxide in the mitochondria of live cells and was validated for confocal microscopy and flow cytometry. This protocol describes simultaneous measurements of mitochondrial superoxide generation with apoptotic markers (Annexin V and Sytox Green) by both flow cytometry and confocal microscopy in endothelial cell lines. The advantages of the described flow cytometry method over other cell-based techniques are the tremendous speed (1–2 h), exquisite precision and the possibility of simultaneous quantitative measurements of mitochondrial superoxide generation and apoptotic (and other) markers, with maximal preservation of cellular functions. This method combined with fluorescent microscopy may be very useful to reveal important spatial–temporal changes in mitochondrial superoxide production and execution of programmed cell death in virtually any cell type.
The discovery that the free radical superoxide anion is a biologically significant molecule has stimulated a remarkable momentum for scientific research in all the fields of biology and medicine1. Emerging recent evidence (based mostly on experiments derived from isolated mitochondria) suggests that under various pathological conditions (e.g., neurodegenerative and inflammatory disorders, diabetes, diabetic complications, cancer and aging) mitochondria may emerge as a significant site of superoxide and other reactive oxygen and nitrogen species generation, in addition to the cytosolic xanthine and NADPH oxidases2–5. The recognition of the crucial role of the mitochondria in the pathological generation of reactive oxygen species in disease rekindled significant interest in the development of methods to assess the mitochondrial superoxide generation, which was largely hindered by the lack of a sensitive and specific assay6–8. Hydroethidine (HE) has been widely used to detect intracellular superoxide anion. The oxidation of HE by superoxide leads to the fluorescent product 2-hydroxy-ethidium9,10, which is excitable at 480 nm wavelength, with emission maximum at 567 nm9,10. Recently, a novel fluoroprobe, a derivative of HE, MitoSOX Red was introduced for selective detection of superoxide in the mitochondria of live cells. The positive charge on the phosphonium group in MitoSOX Red selectively targets this cell-permeant HE derivative to mitochondria, where it accumulates as a function of mitochondrial membrane potential and exhibits fluorescence upon oxidation and subsequent binding to mitochondrial DNA7. In numerous recent studies, MitoSOX was validated with fluorescent/confocal microscopy7,11–19 and flow cytometry19–25 for selective detection of mitochondrial superoxide production in endothelial cells18,19,25, cardiomyocytes16,19,22,25, keratinocytes11, fibroblasts17, epithelial and lymphoid cells20,21,23,24, and neuronal cells7,12–15, to name just a few (see also Table 1 for more details), as well as in isolated vessels26.
Annexin Vand Sytox Green are widely used markers to evaluate apoptosis in various cell types using confocal microscopy and/or flow cytometry27–29. In apoptotic cells, phosphatidylserine is externalized to the plasma membrane surface, which can be measured by flow cytometry and confocal microscopy using phosphatidylserine-binding protein Annexin V conjugated to fluorochromes18,28,29. In conjunction with the permeability probe Sytox Green dye (which works similarly to propidium iodide), a distinction can be made between dying cells with intact plasma membrane integrity and necrotic cells (Sytox Green is impermeant to live or apoptotic cells but stains dead cells with intense green fluorescence by binding to cellular nucleic acids27–29).
As mitochondria play a fundamental role in apoptosis, which can be triggered by increased reactive oxygen and nitrogen species, through mitochondrial membrane permeabilization and release of proapoptotic factors from the mitochondrial intermembrane space to the cytosol, there is considerable interest in cell biology to study the spatial–temporal aspects of changes in mitochondrial superoxide generation and execution of programmed cell death. We have developed a protocol that allows simultaneous measurements of mitochondrial superoxide generation with apoptotic markers (Annexin A5 and/or Sytox Green) by both flow cytometry and confocal microscopy. The advantages of the described flow cytometry method over other cell-based techniques are the tremendous speed, exquisite precision and the possibility of simultaneous quantitative measurements of mitochondrial superoxide generation with apoptosis (or other) markers, with maximal preservation of cellular functions. This method combined with fluorescent microscopy, with careful interpretation of its limitations, may be very useful to reveal important spatial–temporal changes in mitochondrial superoxide production and execution of programmed cell death in virtually any cell type. Although in this protocol we used endothelial cells as a model system to simultaneously study the mitochondrial superoxide production and apoptosis in live cells by flow cytometry and confocal microscopy, this method can easily be adapted for cell types in which the use of MitoSOX was already reported either by confocal microscopy or by flow cytometry (e.g., H9c2 cardiomyocytes19, isolated cardiomyocytes16, human coronary artery and mouse pulmonary microvascular endothelial cells (MPMVECs)18,19,25, motor12, hippocampal14, cortical14 and cerebellar13 neurons, oligodendrocytes7, neuroblastoma cells15, fibroblasts17, human Burkitt’s lymphoma20, human promyelocytic leukemia21, human hepatoma22, colon carcinoma epithelial23 and human esophageal adenocarcinoma cells24, and possibly any other cell types following proper optimization of the loading conditions) (see also Table 1 for reported examples). The use of proper positive controls (e.g., Antimycin A, doxorubicin (DOX), high glucose) and negative controls (e.g., superoxide dismutase mimetics, mitochondrial uncouplers) to validate mitochondrial superoxide generation with MitoSOX in a previously not reported cell type is very important.
FACS binding buffer Hank’s buffered salt solution (GIBCO, Invitrogen) containing 5 mM calcium chloride and magnesium chloride with 1% wt/vol BSA (cell culture tested; Sigma)
MitoSOX Red Store at −20 °C. Dissolve in 13.2 μl DMSO for 5 mM(1,000×) stock just before (<15 min) the experiment. Prepare in the dark and cover with a foil. ! CAUTION Potentially carcinogenic; use nitrile gloves.
Sytox Green nucleic acid stain Supplied as a 5 mM solution in DMSO. Prepare working stock at 1 μM in FACS binding buffer. Store at −20 °C. ! CAUTION Potentially harmful; use nitrile gloves.
Antimycin A Prepare a 10 mM stock in ethyl alcohol. Store at −20 °C. ! CAUTION Highly toxic; use gloves and prepare in a biohazard hood.
DOX Dissolve 1.16 mg in 2 ml water to make 1 mM stock. Store at −20 °C in aliquots. ! CAUTION Toxin and potentially carcinogenic; use gloves.
DETANONOate Dissolve 1.22 mg of DETANONOate in 250 μl water to make 30mM solution (1,000× stock). Prepare just before use. ! CAUTION Potentially hazardous; use gloves.
SOD-PEG Dissolve as 50 U μl−1 stock. Use 500 U per 2 ml media per well. ! CAUTION Potentially skin irritant and hazardous.
ECM 121 mM NaCl, 5 mM NaHCO3, 10 mM Na-HEPES, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2 mM CaCl2, 10 mM glucose and 2.0% dextran, pH 7.4; all obtained from Sigma.
The total amount of time necessary for flow cytometry is about 1–2 h, but may vary, largely depending on the experimental design. The procedure consists of 30 min of MitoSOX Red loading, 15 min for processing the cells, 15 min for Sytox Green/Annexin V-APC binding or Antimycin A treatment, and 15 min for flow cytometry measurement, including control dye standardization. For confocal microscopy, MitoSOX loading requires 30–40 min and Annexin V-APC 15 min. Also considering the warm-up time for the microscope (1 h), acclimatization of cells and image capture, depending on the number of preparations and captured images, the process may take up to 2–8 h.
Step 1A(i): Endothelial cells should not be allowed to grow longer than 48 h before the experiments in plate or dish, because that will prolong trypsinization time, which may interfere with Annexin V binding.
Step 1A(iii): MitoSOX Red loading is dependent on the mitochondrial membrane potential, and any reagents interfering with it should be avoided. Depending on the cell type, the time for MitoSOX Red loading and optimal MitoSOX Red loading concentrations may vary significantly (see also Table 1), and should be optimized individually. Addition of nitric oxide synthase inhibitors may improve the MitoSOX Red intensity in certain cell types in which nitric oxide may form peroxynitrite fast from the mitochondrial superoxide to reduce the MitoSOX Red oxidation.
Step 1A(ix): We observed strong nuclear staining of the MitoSOX Red in some instances due to loss of either mitochondrial structure or membrane potential in dying cells, a limitation that we explain in detail in the next section. For proper comparison among different groups, it is important to analyze multiple markers, including early apoptosis marker Annexin V and cell death dye Sytox Green.
Simultaneous measurement of mitochondrial superoxide generation with apoptotic markers by flow cytometry allows simple, quick and quantitative detection of these markers, with maximal preservation of cellular functions. Importantly, this method also reveals important limitations of the use of MitoSOX Red alone for flow cytometry, without simultaneous measurements of cell death markers, under conditions when a high rate of cell death is expected (see Fig. 1 and below). This method combined with fluorescent microscopy may be very useful to reveal important spatial–temporal changes in mitochondrial superoxide production and execution of programmed cell death in virtually any cell type. Below we describe some experiments we have performed using this protocol, as an example of the results that can be obtained.
First, we measured acute mitochondrial superoxide formation by flow cytometry in HCAECs using only MitoSOX Red under a condition when apoptosis is not significant. Antimycin A rapidly increased mitochondrial superoxide generation in HCAECs measured by both flow cytometry (Fig. 2a) and confocal microscopy (Fig. 2b–d). Oxidized MitoSOX Red (red color) was nicely colocalized with GFP (green color) targeted to the mitochondria (Fig. 2b, colocalization is shown in yellow in overlaid images).
Next, we simultaneously measured mitochondrial apoptotic markers and mitochondrial superoxide formation by flow cytomery in HCAECs using the chemotherapeutic agent DOX, which is known to trigger both a marked increase in mitochondrial superoxide production and cell death (both apoptotic and necrotic). The data in Figure 1a represent the distribution of nonapoptotic, apoptotic and dead cells by Annexin V-APC and Sytox Green staining and histogram analysis of mean intensity of oxidized MitoSOX Red in HCAECs treated with DOX for 16 h. Cells were separated into three groups: nonapoptotic normal cells (light green), early apoptotic cells (blue) and dead cells (orange) (Fig. 1b). As shown in Figure 1a,b, DOX induced a dose-dependent increase in the number of apoptotic and necrotic endothelial cells, as well as in the intensity of MitoSOX fluorescence. However, detailed analysis by flow cytometry of various cell populations, complemented with confocal microscopy, revealed that in dead (orange histograms) cells the oxidized MitoSOX gives very strong fluorescence (Fig. 1a, orange), because of the release of the fluorophore from the mitochondria and binding to nuclear DNA (Fig. 1b, marked cell). Therefore, it is extremely important to exclude the dead cells from the analysis of the mitochondrial superoxide generation (especially if a large number of such cells is expected during the experiment), because the total fluorescence intensity of oxidized MitoSOX Red measured by flow cytometry may be misleading.
The specificity of mitochondrial superoxide production measured by MitoSOX Red in normal cells could be verified by SOD-PEG (a cell-permeable superoxide dismutase that scavenges superoxide; 1,000 U per 2 ml) and DETANONOate (30 μM; a nitric oxide donor that promptly reacts with superoxide to form peroxynitrite) pretreatment, which markedly reduced the intensity of MitoSOX at the FL2 channel (data not shown); furthermore, it could be verified by the use of the superoxide generator system as described7,18,19. The specificity of the superoxide detection by confocal microscopy can further be enhanced by using the dual-wavelength excitation (at 396 and 510 nm) method proposed by Beckman and co-workers7. (The latter method is not feasible for flow cytometry at present, because most flow cytometers cannot use the proposed second low wavelength.)
This research was supported by the Intramural Research Program of NIH/NIAAA (to P.P.). M.M. was supported by AHA (0530087N) and NCRR (ISIORR022511-01A1) grants. B.J.H. was supported by a fellowship from the Hypertension Association. We are indebted to Professors Joseph S. Beckman and Balaraman Kalyanaraman for reading the protocol and for valuable comments.
COMPETING INTERESTS STATEMENT The authors declare no competing financial interests.
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