Monoclonal antibodies were discovered with red-fluorescent Alexa Fluor 594 goat anti-mouse IgG (1:200; Molecular Probes). mitochondrial toxicity and events levels just like those noticed with submaximal activation of AMPA receptors. As opposed to AMPA receptor-mediated insults, calcineurin inhibition or caspase-9 blockade was enough to avoid cell death brought about by both types of kainate receptors. In keeping with these total outcomes, extended glutamate receptor activation in freshly isolated optic nerves triggered selective activation of chromatin and caspase-3 condensation in oligodendrocytes. General, the data presented here indicates that oligodendrocyte death by excitotoxicity is mediated by -independent and caspase-dependent mechanisms. from mitochondria in to the cytosol, which binds to apoptosis protease activating aspect-1 (Apaf-1), forms an oligomeric set up or apoptosome, and therefore activates caspase-9 and eventually caspase-3 (Hengartner, 2000). Various other released mitochondrial protein such as for example Smac/Diablo donate to caspase activation, whereas apoptosis-inducing aspect and endonuclease G may actually kill separately of caspases (Cand et al., 2002). Combination chat between your extrinsic and intrinsic apoptotic pathways takes place at many levels. For example, the former can recruit the latter by the use of intermediates such as Bid (Li et al., 1998). In addition, recent evidence suggests that mitochondria in some instances may act as amplifiers of caspase activity rather than initiators of caspase activation (Lassus et al., 2002; Marsden et al., 2002). Oligodendrocytes also express glutamate receptors (Verkhratsky and Steinh?user, 2000) and, like neurons, are liable to be damaged by excessive glutamate signaling and (Yoshioka et al., 1996; Matute et al., 1997; McDonald et al., 1998). Excitotoxicity in oligodendrocytes is initiated by Ca2+ influx through AMPA receptors and high- and low-affinity kainate receptors (Snchez-Gmez and Matute, 1999; Alberdi et al., 2002). However, the biochemical events downstream of massive Ca2+ entry that lead to oligodendrocyte death have not yet been characterized. In the present study, we investigated the molecular cascades initiated by the activation of AMPA and kainate receptors that ultimately lead to oligodendrocyte death. Our results indicate that excitotoxic insults induce oligodendrocyte death by caspase-dependent and -independent mechanisms. The differential mechanisms involved are receptor specific and depend on the intensity of their activation. Materials and Methods AMPA and cyclothiazide (CTZ) (Tocris Cookson, Bristol, UK), kainate (Sigma, St. Louis, MO). and GYKI53655, kindly supplied by D. Leander (Eli Lilly and Company, Indianapolis, IN), were first dissolved in an equimolar solution of NaOH (AMPA and kainate), ethanol (CTZ), or DMSO (GYKI53655) and were then added to culture medium to achieve the desired final concentration. l-Glutamic acid and CNQX (Sigma) were dissolved directly in the incubating solution. Primary cultures of oligodendrocytes derived from the optic nerves of 12-d-old Sprague Dawley rats, C57BL/6J wild-type mice, and mice transgenic for the gene (Martinou et al., 1994) were obtained as described previously (Barres et al., 1992), with minor modifications (Alberdi et al., 2002). Cells were seeded into 24-well plates bearing 12-mm-diameter coverslips coated with poly-d-lysine (10 g/ml) and maintained at 37C and 5% CO2 in a chemically defined medium (Barres et al., 1992). After 3 d transgene was assayed using PCR (Martinou et al., 1994) and immunocytochemical staining with monoclonal antibodies to the human Bcl-2 protein (Cambridge Research Biochemicals, London, UK). Oligodendrocyte cultures derived from transgenic mice were strongly immunoreactive to these antibodies, whereas those obtained from wild-type mice were only weakly stained. calibration was performed with the successive addition of 10 mm ionomycin and 2 m Tris-50 mm EGTA, pH 8.5. The [Ca2+]i concentration was estimated by the 340/380 ratio method, using a Oligodendrocyte cultures ZL0454 were exposed to AMPA and kainate receptor agonists as above. Thereafter, cells were loaded with 100 nm tetramethylrhodamine ethyl ester (TMRE) and 1 m calcein AM (both from Molecular Probes). Calcein fluorescence, a common agent used to test cell viability, was used here to quantify the number of cells within the reading field. Fluorescence was measured using a Fluoroskan Ascent plate fluorimeter (Thermo Lab Systems, Altrincham, UK), and data were expressed as a percentage of TMRE/calcein fluorescence in controls. Excitation and emission wavelengths for TMRE and calcein were as suggested by the supplier. All experiments (= 5) were performed at least in triplicate and plotted as mean SEM. Oligodendroglial cultures were exposed to AMPA and kainate receptor agonists as described. Subsequently, cells were loaded with 40 m dichloro-H2-fluorescein diacetate (DCFDA) (Molecular Probes) and 5 g/ml Hoechst 33258 (control dye) to assay the levels of free radicals; loading with 50 m monochlorobimane.Subsequently, cells were loaded with 40 m dichloro-H2-fluorescein diacetate (DCFDA) (Molecular Probes) and 5 g/ml Hoechst 33258 (control dye) to assay the levels of free radicals; loading with 50 m monochlorobimane (MclBim) (Molecular Probes) and 1 m calcein AM (control dye) permitted an evaluation of the levels of reduced glutathione. activation of AMPA receptors. In contrast to AMPA receptor-mediated insults, calcineurin inhibition or caspase-9 blockade was sufficient to prevent cell death triggered by both types of kainate receptors. Consistent with these results, prolonged glutamate receptor activation in freshly isolated optic nerves caused selective activation of caspase-3 and chromatin condensation in oligodendrocytes. Overall, the evidence presented here indicates that oligodendrocyte death by excitotoxicity is mediated by caspase-dependent and -independent mechanisms. from mitochondria into the cytosol, which in turn binds to apoptosis protease activating factor-1 (Apaf-1), forms an oligomeric assembly or apoptosome, and thus activates caspase-9 and subsequently caspase-3 (Hengartner, 2000). Other released mitochondrial proteins such as Smac/Diablo contribute to caspase activation, whereas apoptosis-inducing factor and endonuclease G appear to kill independently of caspases (Cand et al., 2002). Cross talk between the extrinsic and intrinsic apoptotic pathways occurs at several levels. For example, the former can recruit the second option by the use of intermediates such as Bid (Li et al., 1998). In addition, recent evidence suggests that mitochondria in some instances may act as amplifiers of caspase activity rather than initiators of caspase activation (Lassus et al., 2002; Marsden et al., 2002). Oligodendrocytes also express glutamate receptors (Verkhratsky and Steinh?user, 2000) and, like neurons, are liable to be damaged by excessive glutamate signaling and (Yoshioka et al., 1996; Matute et al., 1997; McDonald et al., 1998). Excitotoxicity in oligodendrocytes is initiated by Ca2+ influx through AMPA receptors and high- and low-affinity kainate receptors (Snchez-Gmez and Matute, 1999; Alberdi et al., 2002). However, the biochemical events downstream of massive Ca2+ access that lead to oligodendrocyte death have not yet been characterized. In the present study, we investigated the molecular cascades initiated from the activation of AMPA and kainate receptors that ultimately lead to oligodendrocyte death. Our results indicate that excitotoxic insults induce oligodendrocyte death by caspase-dependent and -self-employed mechanisms. The differential mechanisms involved are receptor specific and depend within the intensity of their activation. Materials and Methods AMPA and cyclothiazide (CTZ) (Tocris Cookson, Bristol, UK), kainate (Sigma, St. Louis, MO). and GYKI53655, kindly supplied by D. Leander (Eli Lilly and Organization, Indianapolis, IN), were first dissolved in an equimolar remedy of NaOH (AMPA and kainate), ethanol (CTZ), or DMSO (GYKI53655) and were then added to tradition medium to achieve the desired final concentration. l-Glutamic acid and CNQX (Sigma) were dissolved directly in the incubating remedy. Primary ethnicities of oligodendrocytes derived from the optic nerves of 12-d-old Sprague Dawley rats, C57BL/6J wild-type mice, and mice transgenic for the gene (Martinou et al., 1994) were obtained as explained previously (Barres et al., 1992), with small modifications (Alberdi et al., 2002). Cells were seeded into 24-well plates bearing 12-mm-diameter coverslips coated with poly-d-lysine (10 g/ml) and managed at 37C and 5% CO2 inside a chemically defined medium (Barres et al., 1992). After 3 d transgene was assayed using PCR (Martinou et al., 1994) and immunocytochemical staining with monoclonal antibodies to the human being Bcl-2 protein (Cambridge Study Biochemicals, London, UK). Oligodendrocyte ethnicities derived from transgenic mice were strongly immunoreactive to these antibodies, whereas those from wild-type mice were only weakly stained. calibration was performed with the successive addition of 10 mm ionomycin and 2 m Tris-50 mm EGTA, pH 8.5. The [Ca2+]i concentration was estimated from the 340/380 percentage method, using a Oligodendrocyte ethnicities were exposed to AMPA and kainate receptor agonists as above. Thereafter, cells were loaded with 100 nm tetramethylrhodamine ethyl ester (TMRE) and 1 m calcein AM (both from Molecular Probes). Calcein fluorescence, a common agent used to test cell viability, was used here to quantify the number of cells within the reading field. Fluorescence was measured using a Fluoroskan Ascent plate fluorimeter (Thermo Lab Systems, Altrincham, UK), and data were expressed as a percentage of TMRE/calcein fluorescence in settings. Excitation and emission wavelengths for TMRE and calcein were as suggested from the supplier. All experiments (= 5) were performed at least in triplicate and plotted as mean SEM. Oligodendroglial ethnicities were exposed to AMPA and kainate receptor agonists as explained. Subsequently, cells were loaded with 40 m dichloro-H2-fluorescein diacetate (DCFDA) (Molecular Probes) and 5 g/ml Hoechst 33258 (control dye) to assay the levels of free radicals; loading with 50 m monochlorobimane (MclBim) (Molecular Probes) and 1 m calcein AM (control dye) permitted an evaluation of the levels of reduced.are supported from the Gobierno Vasco. with submaximal activation of AMPA receptors. In contrast to AMPA receptor-mediated insults, calcineurin inhibition or caspase-9 blockade was adequate to prevent cell death induced by both types of kainate receptors. Consistent with these results, long term glutamate receptor activation in freshly isolated optic nerves caused selective activation of caspase-3 and chromatin condensation in oligodendrocytes. Overall, the evidence offered here shows that oligodendrocyte death by excitotoxicity is definitely mediated by caspase-dependent and -self-employed mechanisms. from mitochondria into the cytosol, which in turn binds to apoptosis protease activating element-1 (Apaf-1), forms an oligomeric assembly or apoptosome, and thus activates caspase-9 and consequently caspase-3 (Hengartner, 2000). Additional released mitochondrial proteins such as Smac/Diablo contribute to caspase activation, whereas apoptosis-inducing element and endonuclease G appear to kill individually of caspases (Cand et al., 2002). Mix talk between the extrinsic and intrinsic apoptotic pathways happens at several levels. For example, the former can recruit the second option by the use of intermediates such as Bid (Li et al., 1998). In addition, recent evidence suggests that mitochondria in some instances may act as amplifiers of caspase activity rather than initiators of caspase activation (Lassus et al., 2002; Marsden et al., 2002). Oligodendrocytes also express glutamate receptors (Verkhratsky and Steinh?user, 2000) and, like neurons, are liable to be damaged by excessive glutamate signaling and (Yoshioka et al., 1996; Matute et al., 1997; McDonald et al., 1998). Excitotoxicity in oligodendrocytes is initiated by Ca2+ influx through AMPA receptors and high- and low-affinity kainate receptors (Snchez-Gmez and Matute, 1999; Alberdi et al., 2002). However, the biochemical events downstream of massive Ca2+ access that lead to oligodendrocyte death have not yet been characterized. In the present study, we investigated the molecular cascades initiated by the activation of AMPA and kainate receptors that ultimately lead to oligodendrocyte death. Our results indicate that excitotoxic insults induce oligodendrocyte death by caspase-dependent and -impartial mechanisms. The differential mechanisms involved are receptor specific and depend around the intensity of their activation. Materials and Methods AMPA and cyclothiazide (CTZ) (Tocris Cookson, Bristol, UK), kainate (Sigma, St. Louis, MO). and GYKI53655, kindly supplied by D. Leander (Eli Lilly and Organization, Indianapolis, IN), were first dissolved in an equimolar answer of NaOH (AMPA and kainate), ethanol (CTZ), or DMSO (GYKI53655) and were then added to culture medium to achieve the desired final concentration. l-Glutamic acid and CNQX (Sigma) were dissolved directly in the incubating answer. Primary cultures of oligodendrocytes derived from the optic nerves of 12-d-old Sprague Dawley rats, C57BL/6J wild-type mice, and mice transgenic for the gene (Martinou et al., 1994) were obtained as explained previously (Barres et al., 1992), with minor modifications (Alberdi et al., 2002). Cells were seeded into 24-well plates bearing 12-mm-diameter coverslips coated with poly-d-lysine (10 g/ml) and managed at 37C and 5% CO2 in a chemically defined medium (Barres et al., 1992). After 3 d transgene was assayed using PCR (Martinou et al., 1994) and immunocytochemical staining with monoclonal antibodies to the human Bcl-2 protein (Cambridge Research Biochemicals, London, UK). Oligodendrocyte cultures derived from transgenic mice were strongly immunoreactive to these antibodies, whereas those obtained from wild-type mice were only weakly stained. calibration was performed with the successive addition of 10 mm ionomycin and 2 m Tris-50 mm EGTA, pH 8.5. The [Ca2+]i concentration was estimated by the 340/380 ratio method, using a Oligodendrocyte cultures were exposed to AMPA and kainate receptor agonists as above. Thereafter, cells were loaded with 100 nm tetramethylrhodamine ethyl ester (TMRE) and 1 m calcein AM (both from Molecular Probes). Calcein fluorescence, a common agent used to test cell viability, was used here to quantify the number of cells within the reading field. Fluorescence was measured using a Fluoroskan Ascent plate fluorimeter (Thermo Lab Systems, Altrincham, UK), and data were expressed as a percentage of TMRE/calcein fluorescence in controls. Excitation and emission wavelengths for TMRE and calcein were as suggested by the supplier. All experiments (= 5) were performed at least in triplicate and plotted as mean SEM. Oligodendroglial cultures were exposed to AMPA and kainate receptor agonists as explained. Subsequently, cells were loaded with 40 m dichloro-H2-fluorescein diacetate (DCFDA) (Molecular Probes) and 5 g/ml Hoechst 33258 (control dye) to assay the levels of free radicals; loading with 50 m monochlorobimane (MclBim) (Molecular Probes) and 1 m calcein AM (control dye) permitted an evaluation of the levels of reduced glutathione. Fluorescence was measured in a CytoFluor-2350 system (Millipore, Bedford, MA), and values were plotted as the percentage of DCFDA/Hoechst fluorescence (free radicals) or as the.Monoclonal antibodies were detected with red-fluorescent Alexa Fluor 594 goat anti-mouse IgG (1:200; Molecular Probes). concomitant blockade of caspases 8 and 9. In turn, maximal activation of high- or low-affinity kainate receptors induced mitochondrial events and toxicity levels much like those observed with submaximal activation of AMPA receptors. In contrast to AMPA receptor-mediated insults, calcineurin inhibition or caspase-9 blockade was sufficient to prevent cell death brought on by both types of kainate receptors. Consistent with these results, prolonged glutamate receptor activation in freshly isolated optic nerves caused selective activation of caspase-3 and chromatin condensation in oligodendrocytes. Overall, the evidence offered here indicates that oligodendrocyte death by excitotoxicity is usually mediated by caspase-dependent and -impartial mechanisms. from mitochondria into the cytosol, which in turn binds to apoptosis protease activating factor-1 (Apaf-1), forms an oligomeric assembly or apoptosome, and thus activates caspase-9 and subsequently caspase-3 (Hengartner, 2000). Other released mitochondrial protein such as for example Smac/Diablo donate to caspase activation, whereas apoptosis-inducing element and endonuclease G may actually kill individually of caspases (Cand et al., 2002). Mix talk between your extrinsic and intrinsic apoptotic pathways happens at several amounts. For instance, the previous can recruit the second option through intermediates such as for example Bet (Li et al., 1998). Furthermore, recent evidence shows that mitochondria occasionally may become amplifiers of caspase activity instead of initiators of caspase activation (Lassus et al., 2002; Marsden et al., 2002). Oligodendrocytes also express glutamate receptors (Verkhratsky and Steinh?consumer, 2000) and, want neurons, are prone to end up being damaged by excessive glutamate signaling and (Yoshioka et al., 1996; Matute et al., 1997; McDonald et al., 1998). Excitotoxicity in oligodendrocytes is set up by Ca2+ influx through AMPA receptors and high- and low-affinity kainate receptors (Snchez-Gmez and Matute, 1999; Alberdi et al., 2002). Nevertheless, the biochemical occasions downstream of substantial Ca2+ admittance that result in oligodendrocyte death never have however been characterized. In today’s study, we looked into the molecular cascades initiated from the activation of AMPA and kainate receptors that eventually result in oligodendrocyte loss of life. Our outcomes indicate that excitotoxic insults induce oligodendrocyte loss of life by caspase-dependent and -3rd party systems. The differential systems included are receptor particular and depend for the strength of their activation. Components and Strategies AMPA and cyclothiazide (CTZ) (Tocris Cookson, Bristol, UK), kainate (Sigma, St. Louis, MO). and GYKI53655, kindly given by D. Leander (Eli Lilly and Business, Indianapolis, IN), had been first dissolved within an equimolar option of NaOH (AMPA and kainate), ethanol (CTZ), or DMSO (GYKI53655) and had been then put into tradition medium to attain the preferred final focus. l-Glutamic acidity and ARHGEF2 CNQX (Sigma) had been dissolved straight in the incubating option. Primary ethnicities of oligodendrocytes produced from the optic nerves of 12-d-old Sprague Dawley rats, C57BL/6J wild-type mice, and mice transgenic for the gene (Martinou et al., 1994) had been obtained as referred to previously (Barres et al., 1992), with small adjustments (Alberdi et al., 2002). Cells had been seeded into 24-well plates bearing 12-mm-diameter coverslips covered with poly-d-lysine (10 g/ml) and taken care of at 37C and 5% CO2 inside a chemically described moderate (Barres et al., 1992). After 3 d transgene was assayed using PCR (Martinou et al., 1994) and immunocytochemical staining with monoclonal antibodies towards the human being Bcl-2 proteins (Cambridge Study Biochemicals, London, UK). Oligodendrocyte ethnicities produced from transgenic mice had been highly immunoreactive to these antibodies, whereas those from wild-type mice had been just weakly stained. calibration was performed using the successive addition of 10 mm ionomycin and 2 m Tris-50 mm EGTA, pH 8.5. The [Ca2+]i focus was estimated from the 340/380 percentage method, utilizing a Oligodendrocyte ethnicities had been subjected to AMPA and kainate receptor agonists as above. Thereafter, cells had been packed with 100 nm tetramethylrhodamine ethyl ester (TMRE) and 1 m calcein AM (both from Molecular Probes). Calcein fluorescence, a common agent utilized to check cell viability, was utilized right here to quantify the amount of cells inside the reading field. Fluorescence was assessed utilizing a Fluoroskan Ascent dish fluorimeter (Thermo Laboratory Systems, Altrincham, UK), and data had been expressed as a share of TMRE/calcein fluorescence in settings. Excitation and emission wavelengths for TMRE and calcein had been as suggested from the provider. All tests (= 5) had been performed at least in triplicate and plotted as mean SEM. Oligodendroglial ethnicities had been subjected to AMPA and kainate receptor agonists as referred to. Subsequently, cells had been packed with 40 m dichloro-H2-fluorescein diacetate (DCFDA) (Molecular Probes) and 5 g/ml Hoechst 33258 (control dye) to assay the degrees of free of charge radicals; launching with 50 m monochlorobimane (MclBim) (Molecular Probes) and 1 m calcein AM (control dye) allowed an evaluation from the levels of decreased glutathione. Fluorescence was assessed inside a CytoFluor-2350 program (Millipore, Bedford, MA), and ideals had been plotted as the percentage of DCFDA/Hoechst fluorescence (free of charge radicals) or as the percentage of MClBim/calcein (glutathione) regarding settings. Excitation and.These features weren’t noticed when agonists were applied as well as CNQX or in the lack of Ca2+ in the tradition medium, relative to earlier findings indicating having less excitotoxicity in oligodendrocytes less than these circumstances (Alberdi et al., 2002). caspase-3 and chromatin condensation in oligodendrocytes. General, the evidence shown here shows that oligodendrocyte loss of life by excitotoxicity can be mediated by caspase-dependent and -3rd party systems. from mitochondria in to the cytosol, which binds to apoptosis protease activating element-1 (Apaf-1), forms an oligomeric set up or apoptosome, and therefore activates caspase-9 and consequently caspase-3 (Hengartner, 2000). Additional released mitochondrial protein such as Smac/Diablo contribute to caspase activation, whereas apoptosis-inducing element and endonuclease G appear to kill individually of caspases (Cand et al., 2002). Mix talk between the extrinsic and intrinsic apoptotic pathways happens at several levels. For example, the former can recruit the second option by the use ZL0454 of intermediates such as Bid (Li et al., 1998). In addition, recent evidence suggests that mitochondria in some instances may act as amplifiers of caspase activity rather than initiators of caspase activation (Lassus et al., 2002; Marsden et al., 2002). Oligodendrocytes also express glutamate receptors (Verkhratsky and Steinh?user, 2000) and, like neurons, are liable to be damaged by excessive glutamate signaling and (Yoshioka et al., 1996; Matute et al., 1997; McDonald et al., 1998). Excitotoxicity in oligodendrocytes is initiated by Ca2+ influx through AMPA receptors and high- and low-affinity kainate receptors (Snchez-Gmez and Matute, 1999; Alberdi et al., 2002). However, the biochemical events downstream of massive Ca2+ access that lead to oligodendrocyte death have not yet been characterized. In the present study, we investigated the molecular cascades initiated from the activation of AMPA and kainate receptors that ultimately lead to oligodendrocyte death. Our results indicate that excitotoxic insults induce oligodendrocyte death by caspase-dependent and -self-employed mechanisms. The differential mechanisms involved are receptor specific and depend within the intensity of their activation. Materials and Methods AMPA and cyclothiazide (CTZ) (Tocris Cookson, Bristol, UK), kainate (Sigma, St. Louis, MO). and GYKI53655, kindly supplied by D. Leander (Eli Lilly and Organization, Indianapolis, IN), were first dissolved in an equimolar remedy of NaOH (AMPA and kainate), ethanol (CTZ), or DMSO (GYKI53655) and were then added to tradition medium to achieve the desired final concentration. l-Glutamic acid and CNQX (Sigma) were dissolved directly in the incubating remedy. Primary ethnicities of oligodendrocytes derived from the optic nerves of 12-d-old Sprague Dawley rats, C57BL/6J wild-type mice, and mice transgenic for the gene (Martinou et al., 1994) were obtained as explained previously (Barres et al., 1992), with small modifications (Alberdi et al., 2002). Cells were seeded into 24-well plates bearing 12-mm-diameter coverslips coated with poly-d-lysine (10 g/ml) and managed at 37C and 5% CO2 inside a chemically defined medium (Barres et al., 1992). After 3 d transgene was assayed using PCR (Martinou et al., 1994) and immunocytochemical ZL0454 staining with monoclonal antibodies to the human being Bcl-2 protein (Cambridge Study Biochemicals, London, UK). Oligodendrocyte ethnicities derived from transgenic mice were strongly immunoreactive to these antibodies, whereas ZL0454 those from wild-type mice were only weakly stained. calibration was performed with the successive addition of 10 mm ionomycin and 2 m Tris-50 mm EGTA, pH 8.5. The [Ca2+]i concentration was estimated from the 340/380 percentage method, using a Oligodendrocyte ethnicities were exposed to AMPA and kainate receptor agonists as above. Thereafter, cells were loaded with 100 nm tetramethylrhodamine ethyl ester (TMRE) and 1 m calcein AM (both from Molecular Probes). Calcein fluorescence, a common agent used to test ZL0454 cell viability, was used here to quantify the number of cells within the reading field. Fluorescence was measured using a Fluoroskan Ascent plate fluorimeter (Thermo Lab Systems, Altrincham, UK), and data were expressed as a percentage of TMRE/calcein fluorescence in settings. Excitation and emission wavelengths for TMRE and calcein were as suggested from the supplier. All experiments (= 5) were performed at least in triplicate and plotted as mean SEM. Oligodendroglial ethnicities were exposed to AMPA and kainate receptor agonists as explained. Subsequently, cells were loaded with 40 m dichloro-H2-fluorescein diacetate (DCFDA) (Molecular Probes) and 5 g/ml Hoechst 33258 (control dye) to assay the levels of free radicals; loading with 50 m monochlorobimane (MclBim) (Molecular Probes) and 1.