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    Synonyms o nitrophenyl EGTA Fluorescent EGTA Price 330 00 Quantity 1 mg
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    99
    Thermo Fisher egta
    Modeling VGCC-dependent glutamate miniature release. ( a ) Schematics illustrating VCell simulations. As in Fig. 6c the black dot indicate space taken up by the vesicle in the XY plane 2.5 nm above the active zone; gray circle, vesicle projection on the active zone plane; green dots, assumed positions of Ca 2+ -release sensors, grid 5 nm. ( b ) Examples of average [ Ca 2+ ] transients at release sensors produced by single VGCC openings (for 0.33 ms) for 4 different VGCC-release sensor distances. Insert, corresponding vesicle fusion probabilities. ( c ) Color-coded map showing dependency of vesicle fusion probability p v (Δ t,d ) on VGCC-vesicle distance d and VGCC open-channel duration Δ t . ( d ) Frequency histograms φ (Δ t ) for the durations of spontaneous P/Q-, N-, and R-type channel opening at V rest obtained using the VGCC gating model 12 ( Fig. 5a ). ( e ) Dependencies of vesicle fusion probability p v ( d ) on VGCC-vesicle distance for different VGCC subtypes. ( f ) Frequency histogram ψ ( d ) for the relative VGCC-vesicle distances in the Clustered model (n = 60 simulated active zones). ( g ) Dependency of spontaneous P/Q-, N-, and R-type channel opening on V rest , calculated using the six-state VGCC gating model 12 ( Fig. 5a ). ( h ) Distribution of V rest in cultured hippocampal neurons (mean 71.9 ± 0.7 mV, n = 98 neurons). ( i ) Cumulative fractions of VGCC-mediated mEPSCs and VGCC numbers plotted as functions of the distance from the vesicular release sensor. ~90% of all VGCC-dependent minis are mediated by only ~20% of all VGCCs present in the active zone located within 70 nm of docked vesicles. ( j ) Model predictions for the effects of <t>BAPTA</t> and <t>EGTA</t> on VGCC-dependent mEPSC frequency. Dotted lines, experimental effects of BAPTA-AM and EGTA-AM as estimated in Fig. 4e .
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    Millipore ethylene glycol tetraacetic acid
    Modeling VGCC-dependent glutamate miniature release. ( a ) Schematics illustrating VCell simulations. As in Fig. 6c the black dot indicate space taken up by the vesicle in the XY plane 2.5 nm above the active zone; gray circle, vesicle projection on the active zone plane; green dots, assumed positions of Ca 2+ -release sensors, grid 5 nm. ( b ) Examples of average [ Ca 2+ ] transients at release sensors produced by single VGCC openings (for 0.33 ms) for 4 different VGCC-release sensor distances. Insert, corresponding vesicle fusion probabilities. ( c ) Color-coded map showing dependency of vesicle fusion probability p v (Δ t,d ) on VGCC-vesicle distance d and VGCC open-channel duration Δ t . ( d ) Frequency histograms φ (Δ t ) for the durations of spontaneous P/Q-, N-, and R-type channel opening at V rest obtained using the VGCC gating model 12 ( Fig. 5a ). ( e ) Dependencies of vesicle fusion probability p v ( d ) on VGCC-vesicle distance for different VGCC subtypes. ( f ) Frequency histogram ψ ( d ) for the relative VGCC-vesicle distances in the Clustered model (n = 60 simulated active zones). ( g ) Dependency of spontaneous P/Q-, N-, and R-type channel opening on V rest , calculated using the six-state VGCC gating model 12 ( Fig. 5a ). ( h ) Distribution of V rest in cultured hippocampal neurons (mean 71.9 ± 0.7 mV, n = 98 neurons). ( i ) Cumulative fractions of VGCC-mediated mEPSCs and VGCC numbers plotted as functions of the distance from the vesicular release sensor. ~90% of all VGCC-dependent minis are mediated by only ~20% of all VGCCs present in the active zone located within 70 nm of docked vesicles. ( j ) Model predictions for the effects of <t>BAPTA</t> and <t>EGTA</t> on VGCC-dependent mEPSC frequency. Dotted lines, experimental effects of BAPTA-AM and EGTA-AM as estimated in Fig. 4e .
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    Millipore ethylene glycol tetraacetic acid egta
    Homocysteine induced NETosis is ROS and Ca2 + influx dependent. Freshly isolated peripheral neutrophils were pretreated with either N-acetyl cysteine (1 mM) ( a ) or <t>Diphenyleneiodonium</t> (20 μM) ( b ) or BAPTA (10 μM) ( e ) or <t>EGTA</t> (10 mM) ( f ) or TMB-8 (20 μM) ( g ) for 30 minutes and followed by activation either in presence or absence of homocysteine alone ( a ) or homocysteine (100 μM) IL-6 (25 ng/ml), PMA (20 ng/ml), LPS (2 μg/ml) for three hours. Extent of NETs formation was measured after staining with SYTOX Green (5 μM) in fluorimeter. Data is represented as percentage of maximal SYTOX Green fluorescence. Statistically significant modulation in NETosis is denoted by asterisk ***p
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    Millipore egta am
    Homocysteine induced NETosis is ROS and Ca2 + influx dependent. Freshly isolated peripheral neutrophils were pretreated with either N-acetyl cysteine (1 mM) ( a ) or <t>Diphenyleneiodonium</t> (20 μM) ( b ) or BAPTA (10 μM) ( e ) or <t>EGTA</t> (10 mM) ( f ) or TMB-8 (20 μM) ( g ) for 30 minutes and followed by activation either in presence or absence of homocysteine alone ( a ) or homocysteine (100 μM) IL-6 (25 ng/ml), PMA (20 ng/ml), LPS (2 μg/ml) for three hours. Extent of NETs formation was measured after staining with SYTOX Green (5 μM) in fluorimeter. Data is represented as percentage of maximal SYTOX Green fluorescence. Statistically significant modulation in NETosis is denoted by asterisk ***p
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    egta  (Tocris)
    99
    Tocris egta
    Homocysteine induced NETosis is ROS and Ca2 + influx dependent. Freshly isolated peripheral neutrophils were pretreated with either N-acetyl cysteine (1 mM) ( a ) or <t>Diphenyleneiodonium</t> (20 μM) ( b ) or BAPTA (10 μM) ( e ) or <t>EGTA</t> (10 mM) ( f ) or TMB-8 (20 μM) ( g ) for 30 minutes and followed by activation either in presence or absence of homocysteine alone ( a ) or homocysteine (100 μM) IL-6 (25 ng/ml), PMA (20 ng/ml), LPS (2 μg/ml) for three hours. Extent of NETs formation was measured after staining with SYTOX Green (5 μM) in fluorimeter. Data is represented as percentage of maximal SYTOX Green fluorescence. Statistically significant modulation in NETosis is denoted by asterisk ***p
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    AnaSpec egta am
    Neuronal stimulation triggers glycolysis in response to energy demand from ion pumping. a. Left: Representative trace of Peredox and RCaMP1h lifetimes simultaneously recorded in a DGC from an acute hippocampal slice. The slice was superfused with 2 µM <t>GSK-2837808A</t> for at least 30 min before the experiment, and the LDH inhibitor was kept in the ACSF during the experiment. The ACSF also contained 1 mM <t>EGTA</t> to reinforce Ca 2+ removal in the nominal 0Ca 2+ condition (but [Ca 2+ ] in the control ACSF was accordingly adjusted to a free concentration of 2 mM, as in any other experiment). Effective Ca 2+ removal was confirmed by the absence of a RCaMP1h spike upon stimulation. The Peredox lifetime at baseline, and the metabolic transients in response to neuronal stimulation, were recorded after substituting the bath solution with a 0Ca 2+ ACSF (to obtain Na + -only NADH CYT responses), and the further application of 10 µM α-pompilidotoxin (α-Pmtx, a toxin that prevents voltage-gated Na + channel inactivation). Right: The Na + -only NADH CYT transient was increased in the presence of α-pompilidotoxin. The Peredox lifetime change in response to stimulation was diminished in the absence of Ca 2+ but bounced back to higher amplitudes by increasing Na + influx. The data were compared using a repeated measures ANOVA with a Student-Newman-Keuls post-test (N neurons = 35, N slices = 5 and N mice = 3). For all panels, only neurons showing an initial ΔPeredox lifetime response ≥0.05 ns were included for analysis. b. Representative trace of Peredox and RCaMP1h lifetimes in a DGC stimulated with trains of 100 and 200 electrical pulses. The two-stimulation protocol was also performed in 0Ca 2+ ACSF before and after the application of 3 µM α-pompilidotoxin. The latter condition was followed by the application of the Na + /K + ATPase inhibitor strophanthidin. As in (a) , the slices were exposed to the LDH inhibitor GSK-2837808A from 30 min prior, until the end of the experiment. Likewise, all the solutions contained 1 mM EGTA. c. Comparison of the Peredox lifetime changes in response to both stimulation paradigms (100 or 200 pulses) among the conditions in (b) . The NADH CYT transients in control condition was different from the other conditions (the discontinuous line for the associated p-value applies to all comparisons). The Na + -only NADH CYT responses recorded in 0Ca 2+ ACSF were increased slightly but significantly increased by the application of 3 µM α-pompilidotoxin, an effect that was reversed by strophanthidin. The data were compared using a non-parametric repeated measures ANOVA (Friedman test) with a Dunn post-test (N neurons = 66, N slices = 10 and N mice = 5).
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    92
    Boston BioProducts egta
    Neuronal stimulation triggers glycolysis in response to energy demand from ion pumping. a. Left: Representative trace of Peredox and RCaMP1h lifetimes simultaneously recorded in a DGC from an acute hippocampal slice. The slice was superfused with 2 µM <t>GSK-2837808A</t> for at least 30 min before the experiment, and the LDH inhibitor was kept in the ACSF during the experiment. The ACSF also contained 1 mM <t>EGTA</t> to reinforce Ca 2+ removal in the nominal 0Ca 2+ condition (but [Ca 2+ ] in the control ACSF was accordingly adjusted to a free concentration of 2 mM, as in any other experiment). Effective Ca 2+ removal was confirmed by the absence of a RCaMP1h spike upon stimulation. The Peredox lifetime at baseline, and the metabolic transients in response to neuronal stimulation, were recorded after substituting the bath solution with a 0Ca 2+ ACSF (to obtain Na + -only NADH CYT responses), and the further application of 10 µM α-pompilidotoxin (α-Pmtx, a toxin that prevents voltage-gated Na + channel inactivation). Right: The Na + -only NADH CYT transient was increased in the presence of α-pompilidotoxin. The Peredox lifetime change in response to stimulation was diminished in the absence of Ca 2+ but bounced back to higher amplitudes by increasing Na + influx. The data were compared using a repeated measures ANOVA with a Student-Newman-Keuls post-test (N neurons = 35, N slices = 5 and N mice = 3). For all panels, only neurons showing an initial ΔPeredox lifetime response ≥0.05 ns were included for analysis. b. Representative trace of Peredox and RCaMP1h lifetimes in a DGC stimulated with trains of 100 and 200 electrical pulses. The two-stimulation protocol was also performed in 0Ca 2+ ACSF before and after the application of 3 µM α-pompilidotoxin. The latter condition was followed by the application of the Na + /K + ATPase inhibitor strophanthidin. As in (a) , the slices were exposed to the LDH inhibitor GSK-2837808A from 30 min prior, until the end of the experiment. Likewise, all the solutions contained 1 mM EGTA. c. Comparison of the Peredox lifetime changes in response to both stimulation paradigms (100 or 200 pulses) among the conditions in (b) . The NADH CYT transients in control condition was different from the other conditions (the discontinuous line for the associated p-value applies to all comparisons). The Na + -only NADH CYT responses recorded in 0Ca 2+ ACSF were increased slightly but significantly increased by the application of 3 µM α-pompilidotoxin, an effect that was reversed by strophanthidin. The data were compared using a non-parametric repeated measures ANOVA (Friedman test) with a Dunn post-test (N neurons = 66, N slices = 10 and N mice = 5).
    Egta, supplied by Boston BioProducts, used in various techniques. Bioz Stars score: 92/100, based on 177 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Dojindo Labs egta
    ATP's antimicrobial effect is attributable to its iron-chelating activity. (a) Blocking effects of MgCl 2 and FeCl 3 on ATP's anti- M. intracellulare antimicrobial activity. (b) Anti- M. intracellulare activities of ATP and various metal-chelating agents (pyrophosphate (PPi), <t>EDTA,</t> <t>EGTA)</t> and blocking effects of MgCl 2 . (c) Anti- S. aureus activities of ATP and various metal-chelating agents. (d) Ferric ion-chelating activities of ATP and EDTA measured by CAS assay. (e) Ferric ion-mediated reduction of antimicrobial effects of ATP and EDTA against M. intracellulare . (f) Ferric ion-mediated reduction of antimicrobial effects of ATP and EDTA against P. aeruginosa . (g) Siderophore production by ATP-resistant E. coli during the course of cultivation. (h) Siderophore production by two ATP-resistant K. pneumoniae strains during 48-h cultivation in the presence of ATP. (i) Siderophore production by ATP-susceptible S. aureus and ATP-resistant E. coli during 24-h cultivation in the presence of ATP (5 mM). (j) Siderophore production by ATP-susceptible and ATP-resistant strains of P. aeruginosa during 24-h cultivation.
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    Thermo Fisher np egta am
    ATP's antimicrobial effect is attributable to its iron-chelating activity. (a) Blocking effects of MgCl 2 and FeCl 3 on ATP's anti- M. intracellulare antimicrobial activity. (b) Anti- M. intracellulare activities of ATP and various metal-chelating agents (pyrophosphate (PPi), <t>EDTA,</t> <t>EGTA)</t> and blocking effects of MgCl 2 . (c) Anti- S. aureus activities of ATP and various metal-chelating agents. (d) Ferric ion-chelating activities of ATP and EDTA measured by CAS assay. (e) Ferric ion-mediated reduction of antimicrobial effects of ATP and EDTA against M. intracellulare . (f) Ferric ion-mediated reduction of antimicrobial effects of ATP and EDTA against P. aeruginosa . (g) Siderophore production by ATP-resistant E. coli during the course of cultivation. (h) Siderophore production by two ATP-resistant K. pneumoniae strains during 48-h cultivation in the presence of ATP. (i) Siderophore production by ATP-susceptible S. aureus and ATP-resistant E. coli during 24-h cultivation in the presence of ATP (5 mM). (j) Siderophore production by ATP-susceptible and ATP-resistant strains of P. aeruginosa during 24-h cultivation.
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    Fisher Scientific egta
    Typical force versus relative deformation profiles for <t>MDA-MB-468</t> cells labeled with various dyes: a control cell (1, red), a <t>EGTA</t> treated cell (2, gray), and cells labeled with 5 μM CFDA-SE (3, blue), CMFDA (4, green), CMTMR (5, orange) and Calcein
    Egta, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 92/100, based on 299 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck & Co egta
    Protoplast shrinkage at <t>55°C</t> HS is an ATP- and Ca 2+ -independent process. A Mitochondria in BY-2 cells stained with MitoTracker Red and imaged 10 min after 55°C or 85°C HS, or after treatment with 48 µM CCCP. Severely damaged mitochondria were observed upon all three treatments. B Loss of intracellular ATP content upon HS. Snap freeze-thaw treatment in liquid nitrogen (N 2 ) and CCCP treatment were used as positive controls for completely disrupted and uncoupled mitochondria, respectively. The experiment was repeated twice, each time using four biological replicates per treatment. C MitoTracker Red staining of BY-2 cells exposed to 55°C in the presence or absence of 15 µM Cyclosporin A (CsA) reveals that inhibition of MPTP opening does not rescue mitochondria from severe damage and loss of membrane potential caused by HS. D MitoTracker Red localization in the cells pre-treated with 10 mM <t>EGTA</t> prior to the HS reveals that chelation of extracellular Ca 2+ does not rescue mitochondrial phenotype. E, F Dynamicsof cell death (% SO-positive cells; E ) and protoplast shrinkage (F) in cells with normal and uncoupled (48 µM CCCP treatment) mitochondria. G, H Pre-treatment with 10 mM EGTA before HS does not affect dynamics of cell death (% SO-positive cells; G ) and protoplast shrinkage (H) . Experiments shown in E-H were repeated three times, with ≥ 184 cells per treatment and time point. Each microscopy experiment was performed at least twice. Scale bars, 20 µm ( A ) or 50 µm ( C, D ). IQR, interquartile range. B, E - H , one-way ANOVA with Dunnet’s test; *, p
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    Thermo Fisher egta tetra acetoxymethyl ester egta am
    Protoplast shrinkage at <t>55°C</t> HS is an ATP- and Ca 2+ -independent process. A Mitochondria in BY-2 cells stained with MitoTracker Red and imaged 10 min after 55°C or 85°C HS, or after treatment with 48 µM CCCP. Severely damaged mitochondria were observed upon all three treatments. B Loss of intracellular ATP content upon HS. Snap freeze-thaw treatment in liquid nitrogen (N 2 ) and CCCP treatment were used as positive controls for completely disrupted and uncoupled mitochondria, respectively. The experiment was repeated twice, each time using four biological replicates per treatment. C MitoTracker Red staining of BY-2 cells exposed to 55°C in the presence or absence of 15 µM Cyclosporin A (CsA) reveals that inhibition of MPTP opening does not rescue mitochondria from severe damage and loss of membrane potential caused by HS. D MitoTracker Red localization in the cells pre-treated with 10 mM <t>EGTA</t> prior to the HS reveals that chelation of extracellular Ca 2+ does not rescue mitochondrial phenotype. E, F Dynamicsof cell death (% SO-positive cells; E ) and protoplast shrinkage (F) in cells with normal and uncoupled (48 µM CCCP treatment) mitochondria. G, H Pre-treatment with 10 mM EGTA before HS does not affect dynamics of cell death (% SO-positive cells; G ) and protoplast shrinkage (H) . Experiments shown in E-H were repeated three times, with ≥ 184 cells per treatment and time point. Each microscopy experiment was performed at least twice. Scale bars, 20 µm ( A ) or 50 µm ( C, D ). IQR, interquartile range. B, E - H , one-way ANOVA with Dunnet’s test; *, p
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    Carl Roth GmbH egta
    Effect of PF-IgGs on binding of Dsg1-coated beads to <t>HaCaT</t> cells. Beads were allowed to settle on the surface of HaCaT cells for 30 minutes (control). Number of bound beads was reduced by simultaneous incubation of <t>EGTA</t> (5 mM, 30 minutes). Incubation of monolayers with attached beads for an additional 30 minutes with IgG fractions from 2 patients (PF1- and PF2-IgG, 35 μg/ml each) as well as with a monoclonal antibody (1:50) directed against the extracellular domain of human Dsg1 significantly reduced the number of bound beads (label in white box and white bars). Immunoabsorption using Dsg1-coated beads but not control absorption using VE-cadherin–labeled beads completely abolished the effect of PF-IgGs on bead adhesion. Preincubation of HaCaT cells with PF-IgGs prior to bead settlement also reduced bead binding (label in gray box and gray bars) whereas preincubation of beads with PF-IgGs did not inhibit bead binding. In contrast, the monoclonal Dsg1 antibody reduced bead binding also when applied for preincubation with beads, indicating a different mechanism underlying the reduction of Dsg1 adhesion ( n = 6 for each condition).
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    Millipore egta ethylene glycol bis 2 aminoethylether n
    Effect of PF-IgGs on binding of Dsg1-coated beads to <t>HaCaT</t> cells. Beads were allowed to settle on the surface of HaCaT cells for 30 minutes (control). Number of bound beads was reduced by simultaneous incubation of <t>EGTA</t> (5 mM, 30 minutes). Incubation of monolayers with attached beads for an additional 30 minutes with IgG fractions from 2 patients (PF1- and PF2-IgG, 35 μg/ml each) as well as with a monoclonal antibody (1:50) directed against the extracellular domain of human Dsg1 significantly reduced the number of bound beads (label in white box and white bars). Immunoabsorption using Dsg1-coated beads but not control absorption using VE-cadherin–labeled beads completely abolished the effect of PF-IgGs on bead adhesion. Preincubation of HaCaT cells with PF-IgGs prior to bead settlement also reduced bead binding (label in gray box and gray bars) whereas preincubation of beads with PF-IgGs did not inhibit bead binding. In contrast, the monoclonal Dsg1 antibody reduced bead binding also when applied for preincubation with beads, indicating a different mechanism underlying the reduction of Dsg1 adhesion ( n = 6 for each condition).
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    Amresco egta
    Effects of Ca 2+ and PDE on biophotonic activity in rat retina. A – D Removing intra- and extracellular Ca 2+ together ( A and B , 10 µmol/L <t>BAPTA-AM</t> + 0.5 mmol/L <t>EGTA,</t> n = 5) or introducing a
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    Millipore ethylene glycol bis beta aminoethyl ether n n n n tetraacetic acid tetrasodium salt
    Effects of Ca 2+ and PDE on biophotonic activity in rat retina. A – D Removing intra- and extracellular Ca 2+ together ( A and B , 10 µmol/L <t>BAPTA-AM</t> + 0.5 mmol/L <t>EGTA,</t> n = 5) or introducing a
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    Nacalai egta
    Involvement of intracellular calcium elevation in IL-1α secretion. Macrophages were infected with L. monocytogenes for 1 h, and <t>EGTA</t> or <t>EDTA</t> was added to the cultures at 3 h after infection (A). Alternatively, macrophages were infected with the
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    EGTA Ethylene Glycol Tetraacetic Acid is a useful chemical in a variety of buffers EGTA is a chelating agent that can be used for the determination of calcium in the
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    Image Search Results


    Modeling VGCC-dependent glutamate miniature release. ( a ) Schematics illustrating VCell simulations. As in Fig. 6c the black dot indicate space taken up by the vesicle in the XY plane 2.5 nm above the active zone; gray circle, vesicle projection on the active zone plane; green dots, assumed positions of Ca 2+ -release sensors, grid 5 nm. ( b ) Examples of average [ Ca 2+ ] transients at release sensors produced by single VGCC openings (for 0.33 ms) for 4 different VGCC-release sensor distances. Insert, corresponding vesicle fusion probabilities. ( c ) Color-coded map showing dependency of vesicle fusion probability p v (Δ t,d ) on VGCC-vesicle distance d and VGCC open-channel duration Δ t . ( d ) Frequency histograms φ (Δ t ) for the durations of spontaneous P/Q-, N-, and R-type channel opening at V rest obtained using the VGCC gating model 12 ( Fig. 5a ). ( e ) Dependencies of vesicle fusion probability p v ( d ) on VGCC-vesicle distance for different VGCC subtypes. ( f ) Frequency histogram ψ ( d ) for the relative VGCC-vesicle distances in the Clustered model (n = 60 simulated active zones). ( g ) Dependency of spontaneous P/Q-, N-, and R-type channel opening on V rest , calculated using the six-state VGCC gating model 12 ( Fig. 5a ). ( h ) Distribution of V rest in cultured hippocampal neurons (mean 71.9 ± 0.7 mV, n = 98 neurons). ( i ) Cumulative fractions of VGCC-mediated mEPSCs and VGCC numbers plotted as functions of the distance from the vesicular release sensor. ~90% of all VGCC-dependent minis are mediated by only ~20% of all VGCCs present in the active zone located within 70 nm of docked vesicles. ( j ) Model predictions for the effects of BAPTA and EGTA on VGCC-dependent mEPSC frequency. Dotted lines, experimental effects of BAPTA-AM and EGTA-AM as estimated in Fig. 4e .

    Journal: Nature neuroscience

    Article Title: Differential triggering of spontaneous glutamate release by P/Q-, N-, and R-type Ca2+ channels

    doi: 10.1038/nn.3563

    Figure Lengend Snippet: Modeling VGCC-dependent glutamate miniature release. ( a ) Schematics illustrating VCell simulations. As in Fig. 6c the black dot indicate space taken up by the vesicle in the XY plane 2.5 nm above the active zone; gray circle, vesicle projection on the active zone plane; green dots, assumed positions of Ca 2+ -release sensors, grid 5 nm. ( b ) Examples of average [ Ca 2+ ] transients at release sensors produced by single VGCC openings (for 0.33 ms) for 4 different VGCC-release sensor distances. Insert, corresponding vesicle fusion probabilities. ( c ) Color-coded map showing dependency of vesicle fusion probability p v (Δ t,d ) on VGCC-vesicle distance d and VGCC open-channel duration Δ t . ( d ) Frequency histograms φ (Δ t ) for the durations of spontaneous P/Q-, N-, and R-type channel opening at V rest obtained using the VGCC gating model 12 ( Fig. 5a ). ( e ) Dependencies of vesicle fusion probability p v ( d ) on VGCC-vesicle distance for different VGCC subtypes. ( f ) Frequency histogram ψ ( d ) for the relative VGCC-vesicle distances in the Clustered model (n = 60 simulated active zones). ( g ) Dependency of spontaneous P/Q-, N-, and R-type channel opening on V rest , calculated using the six-state VGCC gating model 12 ( Fig. 5a ). ( h ) Distribution of V rest in cultured hippocampal neurons (mean 71.9 ± 0.7 mV, n = 98 neurons). ( i ) Cumulative fractions of VGCC-mediated mEPSCs and VGCC numbers plotted as functions of the distance from the vesicular release sensor. ~90% of all VGCC-dependent minis are mediated by only ~20% of all VGCCs present in the active zone located within 70 nm of docked vesicles. ( j ) Model predictions for the effects of BAPTA and EGTA on VGCC-dependent mEPSC frequency. Dotted lines, experimental effects of BAPTA-AM and EGTA-AM as estimated in Fig. 4e .

    Article Snippet: Drugs were directly added to the recirculating perfusion system (total volume 10 ml) to achieve the following final concentrations: 0.25 μM ω-Aga (Bachem, Germany), 5 μM ω-Ctx, 0.5 μM SNX 482 (Abcam, UK), 1.0 μM TTA-P2 (Merck, USA), 100 μM CdCl2 (Sigma), 20 μM EGTA-AM and 20 μM BAPTA-AM (Invitrogen, USA).

    Techniques: Produced, Mass Spectrometry, Cell Culture

    Differential effects of slow (EGTA) and fast (BAPTA) exogenous Ca 2+ buffers on VGCC-dependent minis. ( a, b ) Time-course of mEPSC frequency changes during incubation in BAPTA-AM ( a ) and EGTA-AM ( b ). Left, mEPSC traces from representative experiments before and after addition of the Ca 2+ chelators. Right, average responses in N = 7 cells for BAPTA-AM and N = 9 cells for EGTA-AM. ( c, d ) Differential effects of VGCC blockers on mEPSC frequency in BAPTA-AM ( c ) and in EGTA-AM ( d ) pre-treated cultures (both in ( c ) and ( d ) N = 7 cells for ω-Aga and ω-Ctx, and N = 6 cells for ω-Aga, ω-Ctx, and SNX). To determine the remaining fraction of mEPSCs sensitive to VGCC blockade after BAPTA-AM and EGTA-AM treatment (shown on the right), the initial mEPSC frequencies in this set of experiments were normalized to the effects of BAPTA-AM or EGTA-AM determined in ( a ) and ( b ). In contrast to EGTA-AM, pretreatment with BAPTA-AM almost completely occluded the effect of toxins on miniature release. All data are mean ± s.e.m, *** P

    Journal: Nature neuroscience

    Article Title: Differential triggering of spontaneous glutamate release by P/Q-, N-, and R-type Ca2+ channels

    doi: 10.1038/nn.3563

    Figure Lengend Snippet: Differential effects of slow (EGTA) and fast (BAPTA) exogenous Ca 2+ buffers on VGCC-dependent minis. ( a, b ) Time-course of mEPSC frequency changes during incubation in BAPTA-AM ( a ) and EGTA-AM ( b ). Left, mEPSC traces from representative experiments before and after addition of the Ca 2+ chelators. Right, average responses in N = 7 cells for BAPTA-AM and N = 9 cells for EGTA-AM. ( c, d ) Differential effects of VGCC blockers on mEPSC frequency in BAPTA-AM ( c ) and in EGTA-AM ( d ) pre-treated cultures (both in ( c ) and ( d ) N = 7 cells for ω-Aga and ω-Ctx, and N = 6 cells for ω-Aga, ω-Ctx, and SNX). To determine the remaining fraction of mEPSCs sensitive to VGCC blockade after BAPTA-AM and EGTA-AM treatment (shown on the right), the initial mEPSC frequencies in this set of experiments were normalized to the effects of BAPTA-AM or EGTA-AM determined in ( a ) and ( b ). In contrast to EGTA-AM, pretreatment with BAPTA-AM almost completely occluded the effect of toxins on miniature release. All data are mean ± s.e.m, *** P

    Article Snippet: Drugs were directly added to the recirculating perfusion system (total volume 10 ml) to achieve the following final concentrations: 0.25 μM ω-Aga (Bachem, Germany), 5 μM ω-Ctx, 0.5 μM SNX 482 (Abcam, UK), 1.0 μM TTA-P2 (Merck, USA), 100 μM CdCl2 (Sigma), 20 μM EGTA-AM and 20 μM BAPTA-AM (Invitrogen, USA).

    Techniques: Incubation

    FM dye imaging of action potential-evoked exocytosis reveals similar sensitivity of evoked and VGCC-dependent miniature release to presynaptic Ca 2+ chelation. ( a ) Experimental paradigm (see also Online Methods). ( b ) Example of fluorescence loss in individual synaptic boutons during the time course of a typical control experiment. Arrows: individual boutons examined in ( c ). Scale bar 5 μm. ( c ) FM dye de-staining profiles in boutons 1 and 2; exponential fits of de-staining rates at rest ( k rest ) and during 0.5 Hz action potential stimulation ( k stim ) are shown by red dashed lines. The effective specific action potential-evoked FM dye (SRC1) de-staining rate in each bouton was calculated as k AP = k stim – k rest . ( d ) Effect of BAPTA-AM and EGTA-AM on SRC1 de-staining kinetics. Average de-staining profiles from three representative experiments: Control (black), BAPTA-AM (gray) and EGTA-AM (white) symbols. Each profile is an average of 100–200 boutons. ( e ) Comparison of BAPTA-AM and EGTA-AM relative effects on the rate of action potential-evoked vesicular exocytosis k AP (left 2 bars, BAPTA-AM N = 6 experiments, EGTA-AM N = 5 experiments) and on the frequency of VGCC-dependent minis (right 2 bars). The latter was calculated from electrophysiological mEPSC recordings in Figs. 1 and 3 as detailed in Online Methods. Data are mean ± s.e.m, * P

    Journal: Nature neuroscience

    Article Title: Differential triggering of spontaneous glutamate release by P/Q-, N-, and R-type Ca2+ channels

    doi: 10.1038/nn.3563

    Figure Lengend Snippet: FM dye imaging of action potential-evoked exocytosis reveals similar sensitivity of evoked and VGCC-dependent miniature release to presynaptic Ca 2+ chelation. ( a ) Experimental paradigm (see also Online Methods). ( b ) Example of fluorescence loss in individual synaptic boutons during the time course of a typical control experiment. Arrows: individual boutons examined in ( c ). Scale bar 5 μm. ( c ) FM dye de-staining profiles in boutons 1 and 2; exponential fits of de-staining rates at rest ( k rest ) and during 0.5 Hz action potential stimulation ( k stim ) are shown by red dashed lines. The effective specific action potential-evoked FM dye (SRC1) de-staining rate in each bouton was calculated as k AP = k stim – k rest . ( d ) Effect of BAPTA-AM and EGTA-AM on SRC1 de-staining kinetics. Average de-staining profiles from three representative experiments: Control (black), BAPTA-AM (gray) and EGTA-AM (white) symbols. Each profile is an average of 100–200 boutons. ( e ) Comparison of BAPTA-AM and EGTA-AM relative effects on the rate of action potential-evoked vesicular exocytosis k AP (left 2 bars, BAPTA-AM N = 6 experiments, EGTA-AM N = 5 experiments) and on the frequency of VGCC-dependent minis (right 2 bars). The latter was calculated from electrophysiological mEPSC recordings in Figs. 1 and 3 as detailed in Online Methods. Data are mean ± s.e.m, * P

    Article Snippet: Drugs were directly added to the recirculating perfusion system (total volume 10 ml) to achieve the following final concentrations: 0.25 μM ω-Aga (Bachem, Germany), 5 μM ω-Ctx, 0.5 μM SNX 482 (Abcam, UK), 1.0 μM TTA-P2 (Merck, USA), 100 μM CdCl2 (Sigma), 20 μM EGTA-AM and 20 μM BAPTA-AM (Invitrogen, USA).

    Techniques: Imaging, Fluorescence, Staining

    Modeling action potential-evoked release in small hippocampal synapses. ( a ) Allosteric model of Ca 2+ activation of vesicle fusion 19 . ( b ) Presynaptic bouton geometry used in VCell simulations. Scale bar 0.5 μm. ( c ) Representative distributions of VGCCs and vesicles in the active zone for Clustered (left) and Random (right) models. Top, XY cross-sections 2.5 nm above the active zone; bottom, XZ cross-sections corresponding to black dashed lines in the XY plane. Blue dashed lines, active zone borders; brown dots, VGCCs; black dots, space occupied by vesicles; gray circles, vesicle projections on the XY plane; green dots, locations of Ca 2+ -release sensors, grid 5 nm. ( d ) Simulation results corresponding to geometries in ( c ). Top, action potential waveform; middle, average [ Ca 2+ ] transients at Ca 2+ -release sensors; bottom, corresponding release rates; legends, resulting fusion probabilities p v . ( e ) Cumulative probability plots of p v for Clustered and Random models. (n = 28 vesicles from 7 simulated synapses for each model). ( f ) Cumulative probability plots showing the average number of VGCCs located within a given distance from the vesicular Ca 2+ -release sensors (n = 240 vesicles from 60 simulated synapses). ( g ) Model predictions for inhibition of evoked release by BAPTA and EGTA. Dotted lines show the experimental effects of BAPTA-AM and EGTA-AM as determined in Fig. 4e . ( h ) Dependency of p v on Δ[ Ca 2+ ] total simulated by progressive deletion of active VGCCs. Data are from 5 simulated synapses, each point represents average p v for 4 release-ready vesicles. Data on both axes are normalized to the corresponding maximal values at basal conditions. Dotted lines, fitted power function, with the slope corresponding to Ca 2+ current cooperativity m ICa = 2.46.

    Journal: Nature neuroscience

    Article Title: Differential triggering of spontaneous glutamate release by P/Q-, N-, and R-type Ca2+ channels

    doi: 10.1038/nn.3563

    Figure Lengend Snippet: Modeling action potential-evoked release in small hippocampal synapses. ( a ) Allosteric model of Ca 2+ activation of vesicle fusion 19 . ( b ) Presynaptic bouton geometry used in VCell simulations. Scale bar 0.5 μm. ( c ) Representative distributions of VGCCs and vesicles in the active zone for Clustered (left) and Random (right) models. Top, XY cross-sections 2.5 nm above the active zone; bottom, XZ cross-sections corresponding to black dashed lines in the XY plane. Blue dashed lines, active zone borders; brown dots, VGCCs; black dots, space occupied by vesicles; gray circles, vesicle projections on the XY plane; green dots, locations of Ca 2+ -release sensors, grid 5 nm. ( d ) Simulation results corresponding to geometries in ( c ). Top, action potential waveform; middle, average [ Ca 2+ ] transients at Ca 2+ -release sensors; bottom, corresponding release rates; legends, resulting fusion probabilities p v . ( e ) Cumulative probability plots of p v for Clustered and Random models. (n = 28 vesicles from 7 simulated synapses for each model). ( f ) Cumulative probability plots showing the average number of VGCCs located within a given distance from the vesicular Ca 2+ -release sensors (n = 240 vesicles from 60 simulated synapses). ( g ) Model predictions for inhibition of evoked release by BAPTA and EGTA. Dotted lines show the experimental effects of BAPTA-AM and EGTA-AM as determined in Fig. 4e . ( h ) Dependency of p v on Δ[ Ca 2+ ] total simulated by progressive deletion of active VGCCs. Data are from 5 simulated synapses, each point represents average p v for 4 release-ready vesicles. Data on both axes are normalized to the corresponding maximal values at basal conditions. Dotted lines, fitted power function, with the slope corresponding to Ca 2+ current cooperativity m ICa = 2.46.

    Article Snippet: Drugs were directly added to the recirculating perfusion system (total volume 10 ml) to achieve the following final concentrations: 0.25 μM ω-Aga (Bachem, Germany), 5 μM ω-Ctx, 0.5 μM SNX 482 (Abcam, UK), 1.0 μM TTA-P2 (Merck, USA), 100 μM CdCl2 (Sigma), 20 μM EGTA-AM and 20 μM BAPTA-AM (Invitrogen, USA).

    Techniques: Activation Assay, Inhibition

    Homocysteine induced NETosis is ROS and Ca2 + influx dependent. Freshly isolated peripheral neutrophils were pretreated with either N-acetyl cysteine (1 mM) ( a ) or Diphenyleneiodonium (20 μM) ( b ) or BAPTA (10 μM) ( e ) or EGTA (10 mM) ( f ) or TMB-8 (20 μM) ( g ) for 30 minutes and followed by activation either in presence or absence of homocysteine alone ( a ) or homocysteine (100 μM) IL-6 (25 ng/ml), PMA (20 ng/ml), LPS (2 μg/ml) for three hours. Extent of NETs formation was measured after staining with SYTOX Green (5 μM) in fluorimeter. Data is represented as percentage of maximal SYTOX Green fluorescence. Statistically significant modulation in NETosis is denoted by asterisk ***p

    Journal: Scientific Reports

    Article Title: Elevated homocysteine levels in type 2 diabetes induce constitutive neutrophil extracellular traps

    doi: 10.1038/srep36362

    Figure Lengend Snippet: Homocysteine induced NETosis is ROS and Ca2 + influx dependent. Freshly isolated peripheral neutrophils were pretreated with either N-acetyl cysteine (1 mM) ( a ) or Diphenyleneiodonium (20 μM) ( b ) or BAPTA (10 μM) ( e ) or EGTA (10 mM) ( f ) or TMB-8 (20 μM) ( g ) for 30 minutes and followed by activation either in presence or absence of homocysteine alone ( a ) or homocysteine (100 μM) IL-6 (25 ng/ml), PMA (20 ng/ml), LPS (2 μg/ml) for three hours. Extent of NETs formation was measured after staining with SYTOX Green (5 μM) in fluorimeter. Data is represented as percentage of maximal SYTOX Green fluorescence. Statistically significant modulation in NETosis is denoted by asterisk ***p

    Article Snippet: Diphenyleneiodonium (DPI), Ethylene glycol tetra acetic acid (EGTA), BAPTA-AM (Sigma Chemicals, USA) was used to assess role of NADPH oxidase and Ca2+ influx on NETosis respectively.

    Techniques: Isolation, Activation Assay, Staining, Fluorescence

    Neuronal stimulation triggers glycolysis in response to energy demand from ion pumping. a. Left: Representative trace of Peredox and RCaMP1h lifetimes simultaneously recorded in a DGC from an acute hippocampal slice. The slice was superfused with 2 µM GSK-2837808A for at least 30 min before the experiment, and the LDH inhibitor was kept in the ACSF during the experiment. The ACSF also contained 1 mM EGTA to reinforce Ca 2+ removal in the nominal 0Ca 2+ condition (but [Ca 2+ ] in the control ACSF was accordingly adjusted to a free concentration of 2 mM, as in any other experiment). Effective Ca 2+ removal was confirmed by the absence of a RCaMP1h spike upon stimulation. The Peredox lifetime at baseline, and the metabolic transients in response to neuronal stimulation, were recorded after substituting the bath solution with a 0Ca 2+ ACSF (to obtain Na + -only NADH CYT responses), and the further application of 10 µM α-pompilidotoxin (α-Pmtx, a toxin that prevents voltage-gated Na + channel inactivation). Right: The Na + -only NADH CYT transient was increased in the presence of α-pompilidotoxin. The Peredox lifetime change in response to stimulation was diminished in the absence of Ca 2+ but bounced back to higher amplitudes by increasing Na + influx. The data were compared using a repeated measures ANOVA with a Student-Newman-Keuls post-test (N neurons = 35, N slices = 5 and N mice = 3). For all panels, only neurons showing an initial ΔPeredox lifetime response ≥0.05 ns were included for analysis. b. Representative trace of Peredox and RCaMP1h lifetimes in a DGC stimulated with trains of 100 and 200 electrical pulses. The two-stimulation protocol was also performed in 0Ca 2+ ACSF before and after the application of 3 µM α-pompilidotoxin. The latter condition was followed by the application of the Na + /K + ATPase inhibitor strophanthidin. As in (a) , the slices were exposed to the LDH inhibitor GSK-2837808A from 30 min prior, until the end of the experiment. Likewise, all the solutions contained 1 mM EGTA. c. Comparison of the Peredox lifetime changes in response to both stimulation paradigms (100 or 200 pulses) among the conditions in (b) . The NADH CYT transients in control condition was different from the other conditions (the discontinuous line for the associated p-value applies to all comparisons). The Na + -only NADH CYT responses recorded in 0Ca 2+ ACSF were increased slightly but significantly increased by the application of 3 µM α-pompilidotoxin, an effect that was reversed by strophanthidin. The data were compared using a non-parametric repeated measures ANOVA (Friedman test) with a Dunn post-test (N neurons = 66, N slices = 10 and N mice = 5).

    Journal: bioRxiv

    Article Title: The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle

    doi: 10.1101/2020.11.16.385526

    Figure Lengend Snippet: Neuronal stimulation triggers glycolysis in response to energy demand from ion pumping. a. Left: Representative trace of Peredox and RCaMP1h lifetimes simultaneously recorded in a DGC from an acute hippocampal slice. The slice was superfused with 2 µM GSK-2837808A for at least 30 min before the experiment, and the LDH inhibitor was kept in the ACSF during the experiment. The ACSF also contained 1 mM EGTA to reinforce Ca 2+ removal in the nominal 0Ca 2+ condition (but [Ca 2+ ] in the control ACSF was accordingly adjusted to a free concentration of 2 mM, as in any other experiment). Effective Ca 2+ removal was confirmed by the absence of a RCaMP1h spike upon stimulation. The Peredox lifetime at baseline, and the metabolic transients in response to neuronal stimulation, were recorded after substituting the bath solution with a 0Ca 2+ ACSF (to obtain Na + -only NADH CYT responses), and the further application of 10 µM α-pompilidotoxin (α-Pmtx, a toxin that prevents voltage-gated Na + channel inactivation). Right: The Na + -only NADH CYT transient was increased in the presence of α-pompilidotoxin. The Peredox lifetime change in response to stimulation was diminished in the absence of Ca 2+ but bounced back to higher amplitudes by increasing Na + influx. The data were compared using a repeated measures ANOVA with a Student-Newman-Keuls post-test (N neurons = 35, N slices = 5 and N mice = 3). For all panels, only neurons showing an initial ΔPeredox lifetime response ≥0.05 ns were included for analysis. b. Representative trace of Peredox and RCaMP1h lifetimes in a DGC stimulated with trains of 100 and 200 electrical pulses. The two-stimulation protocol was also performed in 0Ca 2+ ACSF before and after the application of 3 µM α-pompilidotoxin. The latter condition was followed by the application of the Na + /K + ATPase inhibitor strophanthidin. As in (a) , the slices were exposed to the LDH inhibitor GSK-2837808A from 30 min prior, until the end of the experiment. Likewise, all the solutions contained 1 mM EGTA. c. Comparison of the Peredox lifetime changes in response to both stimulation paradigms (100 or 200 pulses) among the conditions in (b) . The NADH CYT transients in control condition was different from the other conditions (the discontinuous line for the associated p-value applies to all comparisons). The Na + -only NADH CYT responses recorded in 0Ca 2+ ACSF were increased slightly but significantly increased by the application of 3 µM α-pompilidotoxin, an effect that was reversed by strophanthidin. The data were compared using a non-parametric repeated measures ANOVA (Friedman test) with a Dunn post-test (N neurons = 66, N slices = 10 and N mice = 5).

    Article Snippet: We prepared stock solutions of calmidazolium (100 mM), E6-berbamine (33 mM), EGTA-AM (100 mM), GSK-2837808A (10 mM), isradipine (50 mM), strophanthidin (500 mM) and UK5099 (20 mM) in DMSO.

    Techniques: Concentration Assay, Mouse Assay

    Spontaneous oscillations in the Peredox signal may occur during the prolonged application of zero Ca 2+ -ACSF, in the presence of EGTA and LDH inhibition. Prolonged exposure to a nominal Ca 2+ -free solution, plus LDH inhibition, caused spontaneous elevations of the Peredox lifetime in 25 ± 13% of the cells in each slice (slices=10, mice=5), contrasting with only ∼3% of cells exposed to a brief exposure as in Figure 3c . The spontaneous elevations in Peredox lifetime occurred at any time after removing Ca 2+ from the ACSF. Although these neurons were not included in the analysis, once the spontaneous transients cleared, the experiment resumed as usual. The continuous presence of 1 mM EGTA and 2 µM GSK-2837808 (LDH inhibitor) is not indicated in the figure for simplicity. In the control ACSF, the [Ca 2+ ] was adjusted accordingly to yield a free concentration of 2 mM.

    Journal: bioRxiv

    Article Title: The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle

    doi: 10.1101/2020.11.16.385526

    Figure Lengend Snippet: Spontaneous oscillations in the Peredox signal may occur during the prolonged application of zero Ca 2+ -ACSF, in the presence of EGTA and LDH inhibition. Prolonged exposure to a nominal Ca 2+ -free solution, plus LDH inhibition, caused spontaneous elevations of the Peredox lifetime in 25 ± 13% of the cells in each slice (slices=10, mice=5), contrasting with only ∼3% of cells exposed to a brief exposure as in Figure 3c . The spontaneous elevations in Peredox lifetime occurred at any time after removing Ca 2+ from the ACSF. Although these neurons were not included in the analysis, once the spontaneous transients cleared, the experiment resumed as usual. The continuous presence of 1 mM EGTA and 2 µM GSK-2837808 (LDH inhibitor) is not indicated in the figure for simplicity. In the control ACSF, the [Ca 2+ ] was adjusted accordingly to yield a free concentration of 2 mM.

    Article Snippet: We prepared stock solutions of calmidazolium (100 mM), E6-berbamine (33 mM), EGTA-AM (100 mM), GSK-2837808A (10 mM), isradipine (50 mM), strophanthidin (500 mM) and UK5099 (20 mM) in DMSO.

    Techniques: Inhibition, Mouse Assay, Concentration Assay

    A major Ca 2+ -dependent component of the NADH CYT also occurs under LDH inhibition. a. Comparison of the ΔPeredox/ΔRCaMP values obtained in the continuous presence of the LDH inhibitor GSK-2837808A, before and after the blockade of voltage gated Ca 2+ channels with a combination of isradipine and cadmium (Isra+Cd 2+ ; N neurons = 13, N slices = 3 and N mice = 3). The slices were exposed to GSK-2837808A for at least 30 min prior to the experiment. LDH inhibition should improve the ability to detect the cytosol-only component of Ca 2+ actions on the NADH CYT transients by increasing the control responses to stimulation, as well as by preventing the potential impact of pyruvate accumulation on these transients due to lower Ca 2+ influx into the mitochondria. The effects of the manipulation on the Peredox baseline and the RCaMP spike are also included for the experiments with or without the LDH inhibitor (the sample size is reported in Figure 3 for the latter). b—c. Comparisons for the application of the cell-permeable Ca 2+ chelator EGTA-AM (N neurons = 15, N slices = 3 and N mice = 3 for experiments with LDHi, sample sizes for the other group as in Figure 3 ), or the removal of Ca 2+ from the ACSF (N neurons = 136, N slices = 22 and N mice = 13 for experiments with LDHi, sample sizes for the other group as in Figure 3 ). For all panels, only neurons showing an initial ΔPeredox lifetime response ≥0.05 ns were included for analysis. A paired Student’s t test was used for comparisons between normally distributed data, or a non-parametric paired Wilcoxon test was used otherwise. LDH inhibition partially rescued the Peredox responses in Isra+Cd 2+ and EGTA-AM, but not in 0Ca 2+ +EGTA, even though some pyruvate accumulation is also expected in the last condition due to less Ca 2+ -dependent pyruvate utilization in the mitochondria during stimulation. We do not have a definitive answer for this difference. It is possible that the Ca 2+ channel blockade or Ca 2+ chelation may not be complete, especially in dendrites, triggering a component of the metabolic responses that propagates to the soma, and is better revealed during LDH inhibition.

    Journal: bioRxiv

    Article Title: The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle

    doi: 10.1101/2020.11.16.385526

    Figure Lengend Snippet: A major Ca 2+ -dependent component of the NADH CYT also occurs under LDH inhibition. a. Comparison of the ΔPeredox/ΔRCaMP values obtained in the continuous presence of the LDH inhibitor GSK-2837808A, before and after the blockade of voltage gated Ca 2+ channels with a combination of isradipine and cadmium (Isra+Cd 2+ ; N neurons = 13, N slices = 3 and N mice = 3). The slices were exposed to GSK-2837808A for at least 30 min prior to the experiment. LDH inhibition should improve the ability to detect the cytosol-only component of Ca 2+ actions on the NADH CYT transients by increasing the control responses to stimulation, as well as by preventing the potential impact of pyruvate accumulation on these transients due to lower Ca 2+ influx into the mitochondria. The effects of the manipulation on the Peredox baseline and the RCaMP spike are also included for the experiments with or without the LDH inhibitor (the sample size is reported in Figure 3 for the latter). b—c. Comparisons for the application of the cell-permeable Ca 2+ chelator EGTA-AM (N neurons = 15, N slices = 3 and N mice = 3 for experiments with LDHi, sample sizes for the other group as in Figure 3 ), or the removal of Ca 2+ from the ACSF (N neurons = 136, N slices = 22 and N mice = 13 for experiments with LDHi, sample sizes for the other group as in Figure 3 ). For all panels, only neurons showing an initial ΔPeredox lifetime response ≥0.05 ns were included for analysis. A paired Student’s t test was used for comparisons between normally distributed data, or a non-parametric paired Wilcoxon test was used otherwise. LDH inhibition partially rescued the Peredox responses in Isra+Cd 2+ and EGTA-AM, but not in 0Ca 2+ +EGTA, even though some pyruvate accumulation is also expected in the last condition due to less Ca 2+ -dependent pyruvate utilization in the mitochondria during stimulation. We do not have a definitive answer for this difference. It is possible that the Ca 2+ channel blockade or Ca 2+ chelation may not be complete, especially in dendrites, triggering a component of the metabolic responses that propagates to the soma, and is better revealed during LDH inhibition.

    Article Snippet: We prepared stock solutions of calmidazolium (100 mM), E6-berbamine (33 mM), EGTA-AM (100 mM), GSK-2837808A (10 mM), isradipine (50 mM), strophanthidin (500 mM) and UK5099 (20 mM) in DMSO.

    Techniques: Inhibition, Mouse Assay

    The rise in [Ca 2+ ] CYT , mainly caused by the activity of high voltage-activated Ca 2+ channels, makes a major contribution to the NADH CYT transients in response to stimulation. a. Left: Representative trace from a DGC expressing Peredox and RCaMP1h. The slice was superfused for ∼20 min with the L-type Ca 2+ channel inhibitor isradipine (Isra, 3 µM), and then stimulated. In the continuous presence of isradipine, 20 µM of CdCl 2 (Cd 2+ , a non-selective blocker of voltage-activated Ca 2+ channels) was added to the ACSF. Inhibition of Ca 2+ influx was evident from the progressive reduction of the stimulus associated RCaMP1h spike. Right: The amplitude of the metabolic responses to stimulation (Peredox lifetime change) mirrored the decrease in the RCaMP spikes ( Figure 3 – Supplement 3a ). The data were compared using a non-parametric repeated measures ANOVA (Friedman test) with a Dunn post-test (N neurons = 86, N slices = 11 and N mice = 6). For all panels, only neurons showing an initial ΔPeredox lifetime response ≥0.05 ns were included for analysis. b. Left: Representative trace of a DGC superfused with EGTA-AM (100 µM), a cell-permeable Ca 2+ chelator. As expected, the stimulus-induced RCaMP transients gradually diminished over time, typically stabilizing after ∼1h of treatment. Right: NADH CYT transients are strongly attenuated after effective Ca 2+ buffering by EGTA-AM ( Figure 3 – Supplement 3b ). The data were compared using a Wilcoxon matched pairs test (N neurons = 45, N slices = 5 and N mice = 5). c. Left: Representative trace for the effect of Ca 2+ removal from the bath solution on the metabolic transients in the cytosol. The cell-impermeant Ca 2+ chelator EGTA (1 mM) was added to the ACSF to reinforce Ca 2+ removal after switching to a nominal 0Ca 2+ solution. A modified control ACSF also contained 1 mM EGTA and an adjusted total [Ca 2+ ] resulting in a free concentration of 2 mM, as in any other control experiment. Effective Ca 2+ removal was confirmed by the absence of a RCaMP1h spike upon stimulation. Right: The NADH CYT transients were diminished in a Ca 2+ -deprived ACSF. The data were compared using a Wilcoxon matched pairs test (N neurons = 31, N slices = 7 and N mice = 6).

    Journal: bioRxiv

    Article Title: The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle

    doi: 10.1101/2020.11.16.385526

    Figure Lengend Snippet: The rise in [Ca 2+ ] CYT , mainly caused by the activity of high voltage-activated Ca 2+ channels, makes a major contribution to the NADH CYT transients in response to stimulation. a. Left: Representative trace from a DGC expressing Peredox and RCaMP1h. The slice was superfused for ∼20 min with the L-type Ca 2+ channel inhibitor isradipine (Isra, 3 µM), and then stimulated. In the continuous presence of isradipine, 20 µM of CdCl 2 (Cd 2+ , a non-selective blocker of voltage-activated Ca 2+ channels) was added to the ACSF. Inhibition of Ca 2+ influx was evident from the progressive reduction of the stimulus associated RCaMP1h spike. Right: The amplitude of the metabolic responses to stimulation (Peredox lifetime change) mirrored the decrease in the RCaMP spikes ( Figure 3 – Supplement 3a ). The data were compared using a non-parametric repeated measures ANOVA (Friedman test) with a Dunn post-test (N neurons = 86, N slices = 11 and N mice = 6). For all panels, only neurons showing an initial ΔPeredox lifetime response ≥0.05 ns were included for analysis. b. Left: Representative trace of a DGC superfused with EGTA-AM (100 µM), a cell-permeable Ca 2+ chelator. As expected, the stimulus-induced RCaMP transients gradually diminished over time, typically stabilizing after ∼1h of treatment. Right: NADH CYT transients are strongly attenuated after effective Ca 2+ buffering by EGTA-AM ( Figure 3 – Supplement 3b ). The data were compared using a Wilcoxon matched pairs test (N neurons = 45, N slices = 5 and N mice = 5). c. Left: Representative trace for the effect of Ca 2+ removal from the bath solution on the metabolic transients in the cytosol. The cell-impermeant Ca 2+ chelator EGTA (1 mM) was added to the ACSF to reinforce Ca 2+ removal after switching to a nominal 0Ca 2+ solution. A modified control ACSF also contained 1 mM EGTA and an adjusted total [Ca 2+ ] resulting in a free concentration of 2 mM, as in any other control experiment. Effective Ca 2+ removal was confirmed by the absence of a RCaMP1h spike upon stimulation. Right: The NADH CYT transients were diminished in a Ca 2+ -deprived ACSF. The data were compared using a Wilcoxon matched pairs test (N neurons = 31, N slices = 7 and N mice = 6).

    Article Snippet: We prepared stock solutions of calmidazolium (100 mM), E6-berbamine (33 mM), EGTA-AM (100 mM), GSK-2837808A (10 mM), isradipine (50 mM), strophanthidin (500 mM) and UK5099 (20 mM) in DMSO.

    Techniques: Activity Assay, Expressing, Inhibition, Mouse Assay, Modification, Concentration Assay

    ATP's antimicrobial effect is attributable to its iron-chelating activity. (a) Blocking effects of MgCl 2 and FeCl 3 on ATP's anti- M. intracellulare antimicrobial activity. (b) Anti- M. intracellulare activities of ATP and various metal-chelating agents (pyrophosphate (PPi), EDTA, EGTA) and blocking effects of MgCl 2 . (c) Anti- S. aureus activities of ATP and various metal-chelating agents. (d) Ferric ion-chelating activities of ATP and EDTA measured by CAS assay. (e) Ferric ion-mediated reduction of antimicrobial effects of ATP and EDTA against M. intracellulare . (f) Ferric ion-mediated reduction of antimicrobial effects of ATP and EDTA against P. aeruginosa . (g) Siderophore production by ATP-resistant E. coli during the course of cultivation. (h) Siderophore production by two ATP-resistant K. pneumoniae strains during 48-h cultivation in the presence of ATP. (i) Siderophore production by ATP-susceptible S. aureus and ATP-resistant E. coli during 24-h cultivation in the presence of ATP (5 mM). (j) Siderophore production by ATP-susceptible and ATP-resistant strains of P. aeruginosa during 24-h cultivation.

    Journal: Scientific Reports

    Article Title: ATP Exhibits Antimicrobial Action by Inhibiting Bacterial Utilization of Ferric Ions

    doi: 10.1038/srep08610

    Figure Lengend Snippet: ATP's antimicrobial effect is attributable to its iron-chelating activity. (a) Blocking effects of MgCl 2 and FeCl 3 on ATP's anti- M. intracellulare antimicrobial activity. (b) Anti- M. intracellulare activities of ATP and various metal-chelating agents (pyrophosphate (PPi), EDTA, EGTA) and blocking effects of MgCl 2 . (c) Anti- S. aureus activities of ATP and various metal-chelating agents. (d) Ferric ion-chelating activities of ATP and EDTA measured by CAS assay. (e) Ferric ion-mediated reduction of antimicrobial effects of ATP and EDTA against M. intracellulare . (f) Ferric ion-mediated reduction of antimicrobial effects of ATP and EDTA against P. aeruginosa . (g) Siderophore production by ATP-resistant E. coli during the course of cultivation. (h) Siderophore production by two ATP-resistant K. pneumoniae strains during 48-h cultivation in the presence of ATP. (i) Siderophore production by ATP-susceptible S. aureus and ATP-resistant E. coli during 24-h cultivation in the presence of ATP (5 mM). (j) Siderophore production by ATP-susceptible and ATP-resistant strains of P. aeruginosa during 24-h cultivation.

    Article Snippet: Special agents The following agents were used: ATP (Sigma Aldrich Co., St. Louis, MO; MP Biomedicals, Solon, OH, USA, Calbiochem Co., La Jolla, CA, and Roche Diagnostic Co., Indianapolis, IN), ADP (Sigma), AMP (Sigma), adenosine (Sigma), benzoylbenzoyl ATP (Sigma), oxidized ATP (Sigma), suramin (Wako, Tokyo, Japan), MIA (methyl isobutyl amiloride, Wako), DIDS (4,4′- Diisothiocyanatostilbene-2,2′-disulfonic acid, Sigma), EDTA (Dojindo, Tokyo, Japan), EGTA (Dojindo), dipyridyl (Sigma), CAS (Chrome Azurol S, Sigma), E. coli S17-1 and pK18mobSacB (kindly provided by Dr. F. Taguchi, Okayama University), Instagene matrix (Bio-Rad, Hercules, CA), KOD-Plus (Toyobo, Osaka, Japan), Wizard SV Gel and PCR cleanup system (Promeg, Madison, WI), Ligation High (Toyobo), Protein Assay Rapid Kit (Wako), [14 C] isoleucine (Moravek Biochemicals, Inc, Brea, CA), [14 C]uracil (Moravek Biochemicals, Inc.), α-defensin-1 (Peptide institute, Inc, Osaka, Japan.), cathepsin G (Sigma), vancomycin (Wako), clarithromycin (Taisho-Toyama Pharmaceutical Co., Tokyo), rifampin (Daiichi Sankyo Co., Tokyo), and ethambutol (Sigma), gatifloxacin (Wako), FLUOS (Sigma), LB medium (Invitrogen, San Diego, CA), M9 medium (prepared by our laboratory), Heart infusion agar (Eiken Chemical Co., Tokyo, Japan), Middlebrook 7H9 medium (Becton Dickinson, Cockeysville, MD), and Middlebrook 7H11 medium (Becton Dickinson).

    Techniques: Activity Assay, Blocking Assay

    Typical force versus relative deformation profiles for MDA-MB-468 cells labeled with various dyes: a control cell (1, red), a EGTA treated cell (2, gray), and cells labeled with 5 μM CFDA-SE (3, blue), CMFDA (4, green), CMTMR (5, orange) and Calcein

    Journal: The journal of physical chemistry. B

    Article Title: Cell tracing dyes significantly change single cell mechanics

    doi: 10.1021/jp8103358

    Figure Lengend Snippet: Typical force versus relative deformation profiles for MDA-MB-468 cells labeled with various dyes: a control cell (1, red), a EGTA treated cell (2, gray), and cells labeled with 5 μM CFDA-SE (3, blue), CMFDA (4, green), CMTMR (5, orange) and Calcein

    Article Snippet: To investigate role of calcium ions in cell stiffening MDA-MB-468 cells were incubated with 5 mM ethylene glycol tetraacetic acid, EGTA (Fisher Scientific, Fairlawn, NJ) for 30 min before compression.

    Techniques: Multiple Displacement Amplification, Labeling

    Protoplast shrinkage at 55°C HS is an ATP- and Ca 2+ -independent process. A Mitochondria in BY-2 cells stained with MitoTracker Red and imaged 10 min after 55°C or 85°C HS, or after treatment with 48 µM CCCP. Severely damaged mitochondria were observed upon all three treatments. B Loss of intracellular ATP content upon HS. Snap freeze-thaw treatment in liquid nitrogen (N 2 ) and CCCP treatment were used as positive controls for completely disrupted and uncoupled mitochondria, respectively. The experiment was repeated twice, each time using four biological replicates per treatment. C MitoTracker Red staining of BY-2 cells exposed to 55°C in the presence or absence of 15 µM Cyclosporin A (CsA) reveals that inhibition of MPTP opening does not rescue mitochondria from severe damage and loss of membrane potential caused by HS. D MitoTracker Red localization in the cells pre-treated with 10 mM EGTA prior to the HS reveals that chelation of extracellular Ca 2+ does not rescue mitochondrial phenotype. E, F Dynamicsof cell death (% SO-positive cells; E ) and protoplast shrinkage (F) in cells with normal and uncoupled (48 µM CCCP treatment) mitochondria. G, H Pre-treatment with 10 mM EGTA before HS does not affect dynamics of cell death (% SO-positive cells; G ) and protoplast shrinkage (H) . Experiments shown in E-H were repeated three times, with ≥ 184 cells per treatment and time point. Each microscopy experiment was performed at least twice. Scale bars, 20 µm ( A ) or 50 µm ( C, D ). IQR, interquartile range. B, E - H , one-way ANOVA with Dunnet’s test; *, p

    Journal: bioRxiv

    Article Title: Apoptosis in plants: from semantic appeal to empirical rejection

    doi: 10.1101/2020.09.26.314583

    Figure Lengend Snippet: Protoplast shrinkage at 55°C HS is an ATP- and Ca 2+ -independent process. A Mitochondria in BY-2 cells stained with MitoTracker Red and imaged 10 min after 55°C or 85°C HS, or after treatment with 48 µM CCCP. Severely damaged mitochondria were observed upon all three treatments. B Loss of intracellular ATP content upon HS. Snap freeze-thaw treatment in liquid nitrogen (N 2 ) and CCCP treatment were used as positive controls for completely disrupted and uncoupled mitochondria, respectively. The experiment was repeated twice, each time using four biological replicates per treatment. C MitoTracker Red staining of BY-2 cells exposed to 55°C in the presence or absence of 15 µM Cyclosporin A (CsA) reveals that inhibition of MPTP opening does not rescue mitochondria from severe damage and loss of membrane potential caused by HS. D MitoTracker Red localization in the cells pre-treated with 10 mM EGTA prior to the HS reveals that chelation of extracellular Ca 2+ does not rescue mitochondrial phenotype. E, F Dynamicsof cell death (% SO-positive cells; E ) and protoplast shrinkage (F) in cells with normal and uncoupled (48 µM CCCP treatment) mitochondria. G, H Pre-treatment with 10 mM EGTA before HS does not affect dynamics of cell death (% SO-positive cells; G ) and protoplast shrinkage (H) . Experiments shown in E-H were repeated three times, with ≥ 184 cells per treatment and time point. Each microscopy experiment was performed at least twice. Scale bars, 20 µm ( A ) or 50 µm ( C, D ). IQR, interquartile range. B, E - H , one-way ANOVA with Dunnet’s test; *, p

    Article Snippet: Four different treatments (all at room temperature) were applied prior to 10-min HS at 55°C: (i) 10 mM EGTA (Merck, E3889), pH 8.0 for 10 min, (ii) 0.1% DMSO for 2 h, (iii) 15 µM cyclosporin A (CsA) for 2 h, and (iv) 10 mM EGTA (applied 10 min prior to HS) and 15 µM CsA (applied 2 h prior to HS).

    Techniques: Staining, Inhibition, Microscopy

    HS-induced cell death is ATP- and Ca 2+ -independent process. A-D Morphology of FDA-stained cells under normal conditions (no HS) and after a 55°C HS. Protoplast shrinkage is denoted by arrows. Pre-treatment with 48 µM CCCP for 10 min (B) , 15 µM CsA for 2 h (C ), or 10 mM EGTA for 10 min ( D ) did not alleviate protoplast shrinkage upon HS. Each treatment was repeated at least twice. E, G Sytox Orange (SO) staining of BY-2 cells heat-shocked for 10 min at 40, 45, or 50°C and imaged after 6 h ( E ) and 24 h ( G ). Pre-treatment with CCCP provided no protection against cell death and protoplast shrinkage at any of the tested HS temperatures. F, H Quantification of cell death (% SO-positive cells) in the samples shown in E and G , respectively. DIC, differential interference contrast microscopy. IQR, interquartile range. Experiments shown in F and H were repeated three times, with ≥ 170 cells per treatment and time point. The data was subjected to one-way ANOVA with Bonferroni correction. Scale bars, 20 µm (A-D) or 100 µm (E, G) .

    Journal: bioRxiv

    Article Title: Apoptosis in plants: from semantic appeal to empirical rejection

    doi: 10.1101/2020.09.26.314583

    Figure Lengend Snippet: HS-induced cell death is ATP- and Ca 2+ -independent process. A-D Morphology of FDA-stained cells under normal conditions (no HS) and after a 55°C HS. Protoplast shrinkage is denoted by arrows. Pre-treatment with 48 µM CCCP for 10 min (B) , 15 µM CsA for 2 h (C ), or 10 mM EGTA for 10 min ( D ) did not alleviate protoplast shrinkage upon HS. Each treatment was repeated at least twice. E, G Sytox Orange (SO) staining of BY-2 cells heat-shocked for 10 min at 40, 45, or 50°C and imaged after 6 h ( E ) and 24 h ( G ). Pre-treatment with CCCP provided no protection against cell death and protoplast shrinkage at any of the tested HS temperatures. F, H Quantification of cell death (% SO-positive cells) in the samples shown in E and G , respectively. DIC, differential interference contrast microscopy. IQR, interquartile range. Experiments shown in F and H were repeated three times, with ≥ 170 cells per treatment and time point. The data was subjected to one-way ANOVA with Bonferroni correction. Scale bars, 20 µm (A-D) or 100 µm (E, G) .

    Article Snippet: Four different treatments (all at room temperature) were applied prior to 10-min HS at 55°C: (i) 10 mM EGTA (Merck, E3889), pH 8.0 for 10 min, (ii) 0.1% DMSO for 2 h, (iii) 15 µM cyclosporin A (CsA) for 2 h, and (iv) 10 mM EGTA (applied 10 min prior to HS) and 15 µM CsA (applied 2 h prior to HS).

    Techniques: Staining, Microscopy

    Effect of PF-IgGs on binding of Dsg1-coated beads to HaCaT cells. Beads were allowed to settle on the surface of HaCaT cells for 30 minutes (control). Number of bound beads was reduced by simultaneous incubation of EGTA (5 mM, 30 minutes). Incubation of monolayers with attached beads for an additional 30 minutes with IgG fractions from 2 patients (PF1- and PF2-IgG, 35 μg/ml each) as well as with a monoclonal antibody (1:50) directed against the extracellular domain of human Dsg1 significantly reduced the number of bound beads (label in white box and white bars). Immunoabsorption using Dsg1-coated beads but not control absorption using VE-cadherin–labeled beads completely abolished the effect of PF-IgGs on bead adhesion. Preincubation of HaCaT cells with PF-IgGs prior to bead settlement also reduced bead binding (label in gray box and gray bars) whereas preincubation of beads with PF-IgGs did not inhibit bead binding. In contrast, the monoclonal Dsg1 antibody reduced bead binding also when applied for preincubation with beads, indicating a different mechanism underlying the reduction of Dsg1 adhesion ( n = 6 for each condition).

    Journal: Journal of Clinical Investigation

    Article Title: Pemphigus foliaceus IgG causes dissociation of desmoglein 1-containing junctions without blocking desmoglein 1 transinteraction

    doi: 10.1172/JCI23475

    Figure Lengend Snippet: Effect of PF-IgGs on binding of Dsg1-coated beads to HaCaT cells. Beads were allowed to settle on the surface of HaCaT cells for 30 minutes (control). Number of bound beads was reduced by simultaneous incubation of EGTA (5 mM, 30 minutes). Incubation of monolayers with attached beads for an additional 30 minutes with IgG fractions from 2 patients (PF1- and PF2-IgG, 35 μg/ml each) as well as with a monoclonal antibody (1:50) directed against the extracellular domain of human Dsg1 significantly reduced the number of bound beads (label in white box and white bars). Immunoabsorption using Dsg1-coated beads but not control absorption using VE-cadherin–labeled beads completely abolished the effect of PF-IgGs on bead adhesion. Preincubation of HaCaT cells with PF-IgGs prior to bead settlement also reduced bead binding (label in gray box and gray bars) whereas preincubation of beads with PF-IgGs did not inhibit bead binding. In contrast, the monoclonal Dsg1 antibody reduced bead binding also when applied for preincubation with beads, indicating a different mechanism underlying the reduction of Dsg1 adhesion ( n = 6 for each condition).

    Article Snippet: Negative controls were performed using Dsg1-coated beads incubated on the surface of HaCaT cells in the presence of 5 mM EGTA (Roth) or in the presence of monoclonal mouse IgG antibody (1:50 in HBSS) directed against Dsg-1 (clone p124; Progen Industries Ltd.).

    Techniques: Binding Assay, Incubation, Labeling

    Effects of Ca 2+ and PDE on biophotonic activity in rat retina. A – D Removing intra- and extracellular Ca 2+ together ( A and B , 10 µmol/L BAPTA-AM + 0.5 mmol/L EGTA, n = 5) or introducing a

    Journal: Neuroscience Bulletin

    Article Title: Biophotons Contribute to Retinal Dark Noise

    doi: 10.1007/s12264-016-0029-6

    Figure Lengend Snippet: Effects of Ca 2+ and PDE on biophotonic activity in rat retina. A – D Removing intra- and extracellular Ca 2+ together ( A and B , 10 µmol/L BAPTA-AM + 0.5 mmol/L EGTA, n = 5) or introducing a

    Article Snippet: A phosphodiesterase 6 (PDE6) inhibitor (Zaprinast, 100 nmol/L, Sigma, St. Louis, MO), BAPTA-AM (10 μmol/L, Molecular Probes, Eugene, OR), and EGTA (0.5 mmol/L, Amresco, Solon, OH) were initially dissolved in DMSO and then diluted to their final concentrations in ACSF or Ringer’s solution.

    Techniques: Activity Assay

    Involvement of intracellular calcium elevation in IL-1α secretion. Macrophages were infected with L. monocytogenes for 1 h, and EGTA or EDTA was added to the cultures at 3 h after infection (A). Alternatively, macrophages were infected with the

    Journal: Infection and Immunity

    Article Title: Listeriolysin O-Dependent Bacterial Entry into the Cytoplasm Is Required for Calpain Activation and Interleukin-1? Secretion in Macrophages Infected with Listeria monocytogenes ▿

    doi: 10.1128/IAI.01143-09

    Figure Lengend Snippet: Involvement of intracellular calcium elevation in IL-1α secretion. Macrophages were infected with L. monocytogenes for 1 h, and EGTA or EDTA was added to the cultures at 3 h after infection (A). Alternatively, macrophages were infected with the

    Article Snippet: EDTA and EGTA were purchased from Nacalai Tesque Inc. (Kyoto, Japan).

    Techniques: Infection