np egta am (Thermo Fisher)

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Name:

NP EGTA AM o Nitrophenyl EGTA AM

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Catalog Number:

n6803

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None

Category:

Labeling Detection Products

Applications:

Calcium Detection|Cell Analysis|Ionic Homeostasis & Signaling|Cell Viability, Proliferation & Function

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Structured Review

Thermo Fisher np egta am

Role of cytoplasmic Ca 2+ in DF-triggered ATP release in ECV304 cells. ( A ) DF exposure triggered ATP accumulation in the medium that was assayed after the stimulation interval given in the text. Baseline and triggered ATP release were measured in different experimental groups. Buffering [Ca 2+ ] i with BAPTA or emptying the Ca 2+ stores with thapsigargin reduced DF-triggered ATP release. Inhibitor concentrations and incubation times are given in the text; inhibitors were absent during stimulation. ( B ) [Ca 2+ ] i dynamics in response to DF exposure, demonstrating transient, steady and oscillatory changes. [Ca 2+ ] i changes are given as increases in <t>fluo-3</t> fluorescence (ΔF, arbitrary units). ( C ) Xestospongin reduced DF-triggered ATP release at 2 μM and completely blocked it at 10 μM; dantrolene and pre-emptying Ca 2+ stores with caffeine also reduced the response. ( D ) Photoactivation of Ca 2+ from <t>NP-EGTA</t> or stimulation of Ca 2+ entry with A23187 triggered ATP release. Prolonging NP-EGTA ester loading to 1 h or increasing the A23187 concentration or exposure time did not trigger ATP responses. * Significantly above the corresponding baseline; # significantly below the control response; a single symbol indicates P

Once the cell permeant NP caged EGTA AM is converted to NP EGTA by intracellular esterases it becomes a photolabile chelator that exhibits a high selectivity for Ca2 upon UV illumination its Kd increases from 80 nM to 1 mM
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Images

1) Product Images from "Intracellular calcium changes trigger connexin 32 hemichannel opening"

Article Title: Intracellular calcium changes trigger connexin 32 hemichannel opening

Journal: The EMBO Journal

doi: 10.1038/sj.emboj.7600908

Figure Legend Snippet: Role of cytoplasmic Ca 2+ in DF-triggered ATP release in ECV304 cells. ( A ) DF exposure triggered ATP accumulation in the medium that was assayed after the stimulation interval given in the text. Baseline and triggered ATP release were measured in different experimental groups. Buffering [Ca 2+ ] i with BAPTA or emptying the Ca 2+ stores with thapsigargin reduced DF-triggered ATP release. Inhibitor concentrations and incubation times are given in the text; inhibitors were absent during stimulation. ( B ) [Ca 2+ ] i dynamics in response to DF exposure, demonstrating transient, steady and oscillatory changes. [Ca 2+ ] i changes are given as increases in fluo-3 fluorescence (ΔF, arbitrary units). ( C ) Xestospongin reduced DF-triggered ATP release at 2 μM and completely blocked it at 10 μM; dantrolene and pre-emptying Ca 2+ stores with caffeine also reduced the response. ( D ) Photoactivation of Ca 2+ from NP-EGTA or stimulation of Ca 2+ entry with A23187 triggered ATP release. Prolonging NP-EGTA ester loading to 1 h or increasing the A23187 concentration or exposure time did not trigger ATP responses. * Significantly above the corresponding baseline; # significantly below the control response; a single symbol indicates P

Techniques Used: Incubation, Fluorescence, Concentration Assay

2) Product Images from "Dynamics of SNARE Assembly and Disassembly during Sperm Acrosomal ExocytosisEnSNAREd by the Sperm Acrosome"

Article Title: Dynamics of SNARE Assembly and Disassembly during Sperm Acrosomal ExocytosisEnSNAREd by the Sperm Acrosome

Journal: PLoS Biology

doi: 10.1371/journal.pbio.0030323

Activation with Rab3A Triggers the Disassembly of SNARE Complexes before Intra-Acrosomal Ca 2+ Efflux Permeabilized spermatozoa were loaded with 10 μM NP-EGTA-AM (NP) for 15 min at 37 °C to chelate intra-acrosomal Ca 2+ . Then 357 nM BoNT/E was added to the system, and AE was initiated by adding 300 nM GTP-γ-S-loaded Rab3A. After 15 min at 37 °C, the toxin was inactivated with 2.5 μM TPEN, and photolysis of NP was induced by UV illumination (hν). The samples were incubated for a further 5 min to promote exocytosis (NP→BoNT/E→Rab3A→TPEN→hν, black bar). At the end of the incubation, sperm were fixed and AE was measured as described in Materials and Methods . Several controls are included (grey bars): background AE in the absence of any stimulation (control); AE stimulated by 10 μM free Ca 2+ (Ca 2+ ) or Rab3A (Rab3A); blockage of Rab3A-triggered exocytosis by BoNT/E (BoNT/E→Rab3A); inhibitory effect of NP-EGTA-AM in the dark (NP→Rab3A→dark) and the recovery upon illumination (NP→Rab3A→hν); and inactivation of the neurotoxin by TPEN (NP→TPEN→BoNT/E→Rab3A→hν). The data were normalized as described in Materials and Methods (mean ± SEM). Statistical analysis is provided in Table S4 .

Figure Legend Snippet: Activation with Rab3A Triggers the Disassembly of SNARE Complexes before Intra-Acrosomal Ca 2+ Efflux Permeabilized spermatozoa were loaded with 10 μM NP-EGTA-AM (NP) for 15 min at 37 °C to chelate intra-acrosomal Ca 2+ . Then 357 nM BoNT/E was added to the system, and AE was initiated by adding 300 nM GTP-γ-S-loaded Rab3A. After 15 min at 37 °C, the toxin was inactivated with 2.5 μM TPEN, and photolysis of NP was induced by UV illumination (hν). The samples were incubated for a further 5 min to promote exocytosis (NP→BoNT/E→Rab3A→TPEN→hν, black bar). At the end of the incubation, sperm were fixed and AE was measured as described in Materials and Methods . Several controls are included (grey bars): background AE in the absence of any stimulation (control); AE stimulated by 10 μM free Ca 2+ (Ca 2+ ) or Rab3A (Rab3A); blockage of Rab3A-triggered exocytosis by BoNT/E (BoNT/E→Rab3A); inhibitory effect of NP-EGTA-AM in the dark (NP→Rab3A→dark) and the recovery upon illumination (NP→Rab3A→hν); and inactivation of the neurotoxin by TPEN (NP→TPEN→BoNT/E→Rab3A→hν). The data were normalized as described in Materials and Methods (mean ± SEM). Statistical analysis is provided in Table S4 .

Techniques Used: Activation Assay, Incubation

3) Product Images from "A cAMP and Ca2+ coincidence detector in support of Ca2+-induced Ca2+ release in mouse pancreatic ? cells"

Article Title: A cAMP and Ca2+ coincidence detector in support of Ca2+-induced Ca2+ release in mouse pancreatic ? cells

Journal: The Journal of Physiology

doi: 10.1113/jphysiol.2005.087510

Sensitization of CICR by Ex-4 and forskolin A, an INS-1 cell loaded with NP-EGTA was bathed in SES containing 11.1 m m glucose. The uncaging of Ca 2+ by UV flash photolysis (UV, arrows) produced a small increase of [Ca 2+ ] i but did not trigger CICR. B, a large transient increase of [Ca 2+ ] i (CICR) was observed when UV flash photolysis was performed in the presence but not the absence of Ex-4 (compare y- axis scale bars of A and B ). C, the action of Ex-4, as illustrated on an expanded time scale. CICR amplitude was determined by subtracting the baseline Ca 2+ concentration from the Ca 2+ concentration measured at the peak of the Ca 2+ transient. Also illustrated are the 50% and 10% threshold values of [Ca 2+ ] i that were used when determining the percentage of cells exhibiting CICR (see Methods). D , sensitization of CICR by the cAMP-elevating agent forskolin (2 μ m ). A and D are from two different cells. B and C are from the same cell.

Figure Legend Snippet: Sensitization of CICR by Ex-4 and forskolin A, an INS-1 cell loaded with NP-EGTA was bathed in SES containing 11.1 m m glucose. The uncaging of Ca 2+ by UV flash photolysis (UV, arrows) produced a small increase of [Ca 2+ ] i but did not trigger CICR. B, a large transient increase of [Ca 2+ ] i (CICR) was observed when UV flash photolysis was performed in the presence but not the absence of Ex-4 (compare y- axis scale bars of A and B ). C, the action of Ex-4, as illustrated on an expanded time scale. CICR amplitude was determined by subtracting the baseline Ca 2+ concentration from the Ca 2+ concentration measured at the peak of the Ca 2+ transient. Also illustrated are the 50% and 10% threshold values of [Ca 2+ ] i that were used when determining the percentage of cells exhibiting CICR (see Methods). D , sensitization of CICR by the cAMP-elevating agent forskolin (2 μ m ). A and D are from two different cells. B and C are from the same cell.

Techniques Used: Produced, Concentration Assay

4) Product Images from "IP3 mediated global Ca2+ signals arise through two temporally and spatially distinct modes of Ca2+ release"

Article Title: IP3 mediated global Ca2+ signals arise through two temporally and spatially distinct modes of Ca2+ release

Journal: eLife

doi: 10.7554/eLife.55008

The suppression of Ca 2+ puffs during global signals does not result because of elevated cytosolic [Ca 2+ ]. ( A,B ) IP 3 -evoked Ca 2+ puffs are not suppressed by prior photorelease of Ca 2+ . ( A ) Traces depict fluorescence ratios (black; ΔF/F 0 ) from WT HEK cells and corresponding SD signals (grey; in arbitrary units, A.U.). Records, from left to right, show responses from individual cells loaded with NP-EGTA (caged Ca 2+ ) that were unstimulated or exposed to increasing UV flash durations (marked by arrows) to photorelease progressively increasing amounts of free Ca 2+ before challenging cells with CCH (100 µM) locally delivered by a puffer pipette when indicated by the bars. Traces are blanked out during the artifact caused by the photolysis flash. ( B ) Data points from traces like those in A show the integral under SD trace (a measure of puff activity) as a ratio of the change in global Ca 2+ signal (ΔF/F 0 ) evoked by CCH. The data are binned in terms of the jump in Cal520 fluorescence (ΔF/F 0 ) evoked by photolysis of caged Ca 2+ . Open symbols are from individual cells, and filled symbols are means for each group (respective n numbers for different bins; 20, 4, 4, 6, 8). Data are normalized with respect to the mean ratio without prior photorelease of Ca 2+ . There was no significant difference between control CCH responses and CCH responses following Ca 2+ jumps (evaluated by Student T-test; p values between 0.17 and 0.66 for the different binned groupings). ( C,D ) Termination of puff activity is unaffected when global cytosolic Ca 2+ signals are attenuated by buffering with EGTA. ( C ) Traces showing the Cal520 fluorescence ratio (ΔF/F 0 ; smooth trace) and SD signal (noisy trace) in response to photoreleased i-IP 3 in a representative WT HEK cell that was incubated with 15 µM EGTA/AM to buffer cytosolic Ca 2+ and attenuate the amplitude of the global Ca 2+ signal. ( D ) Scatter plots show measurements of the SD signal at intervals during the rising phase of global Ca 2+ responses against the magnitude of the global Ca 2+ elevation (ΔF/F 0 ) at that time. Measurements were binned at intervals of (0.1 ΔF/F 0 ) and SD data are normalized to a peak value of 1. Solid circles show mean data from 14 EGTA-loaded cells. For comparison, open circles present data reproduced from Figure 3C showing measurements from 11 control cells that gave fast rising responses to photoreleased i-IP 3 .

Figure Legend Snippet: The suppression of Ca 2+ puffs during global signals does not result because of elevated cytosolic [Ca 2+ ]. ( A,B ) IP 3 -evoked Ca 2+ puffs are not suppressed by prior photorelease of Ca 2+ . ( A ) Traces depict fluorescence ratios (black; ΔF/F 0 ) from WT HEK cells and corresponding SD signals (grey; in arbitrary units, A.U.). Records, from left to right, show responses from individual cells loaded with NP-EGTA (caged Ca 2+ ) that were unstimulated or exposed to increasing UV flash durations (marked by arrows) to photorelease progressively increasing amounts of free Ca 2+ before challenging cells with CCH (100 µM) locally delivered by a puffer pipette when indicated by the bars. Traces are blanked out during the artifact caused by the photolysis flash. ( B ) Data points from traces like those in A show the integral under SD trace (a measure of puff activity) as a ratio of the change in global Ca 2+ signal (ΔF/F 0 ) evoked by CCH. The data are binned in terms of the jump in Cal520 fluorescence (ΔF/F 0 ) evoked by photolysis of caged Ca 2+ . Open symbols are from individual cells, and filled symbols are means for each group (respective n numbers for different bins; 20, 4, 4, 6, 8). Data are normalized with respect to the mean ratio without prior photorelease of Ca 2+ . There was no significant difference between control CCH responses and CCH responses following Ca 2+ jumps (evaluated by Student T-test; p values between 0.17 and 0.66 for the different binned groupings). ( C,D ) Termination of puff activity is unaffected when global cytosolic Ca 2+ signals are attenuated by buffering with EGTA. ( C ) Traces showing the Cal520 fluorescence ratio (ΔF/F 0 ; smooth trace) and SD signal (noisy trace) in response to photoreleased i-IP 3 in a representative WT HEK cell that was incubated with 15 µM EGTA/AM to buffer cytosolic Ca 2+ and attenuate the amplitude of the global Ca 2+ signal. ( D ) Scatter plots show measurements of the SD signal at intervals during the rising phase of global Ca 2+ responses against the magnitude of the global Ca 2+ elevation (ΔF/F 0 ) at that time. Measurements were binned at intervals of (0.1 ΔF/F 0 ) and SD data are normalized to a peak value of 1. Solid circles show mean data from 14 EGTA-loaded cells. For comparison, open circles present data reproduced from Figure 3C showing measurements from 11 control cells that gave fast rising responses to photoreleased i-IP 3 .

Techniques Used: Fluorescence, Transferring, Activity Assay, Incubation

5) Product Images from "Sequential exocytosis of insulin granules is associated with redistribution of SNAP25"

Article Title: Sequential exocytosis of insulin granules is associated with redistribution of SNAP25

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200312033

Exocytic events triggered by photolysis of a caged-Ca 2 + compound. (A) SRB fluorescence image of an islet that had been preloaded with NP-EGTA and fura-2. (B) Fura-2 (Ca 2+ ) images obtained 1.2 s before and after UV irradiation of the islet shown in A. (C) Representative time courses of normalized fura-2 fluorescence (black) and of [Ca 2+ ] i estimated with fura-2FF (blue). Fura-2 fluorescence ( F ) was normalized by the resting fluorescence ( F 0 ), and the trace shown is for the islet in B. (D) Exocytic event (arrow) triggered by UV photolysis of NP-EGTA. Ω Profiles often persisted for tens of seconds. (E) Two examples of time courses of sequential exocytosis (blue and red) and an example of that of solitary exocytosis (black) triggered by photolysis of NP-EGTA. The traces were aligned at the onset of exocytosis. (F) Time courses of the decay of SRB fluorescence associated with exocytic events induced by 20 mM glucose (black and red) or by photolysis of NP-EGTA (green). The traces are averages of > 10 exocytic events, which were aligned at the peak of SRB fluorescence and normalized by the peak intensity. The data correspond to solitary events (black and green) or to the primary event of sequential exocytosis (red).

Figure Legend Snippet: Exocytic events triggered by photolysis of a caged-Ca 2 + compound. (A) SRB fluorescence image of an islet that had been preloaded with NP-EGTA and fura-2. (B) Fura-2 (Ca 2+ ) images obtained 1.2 s before and after UV irradiation of the islet shown in A. (C) Representative time courses of normalized fura-2 fluorescence (black) and of [Ca 2+ ] i estimated with fura-2FF (blue). Fura-2 fluorescence ( F ) was normalized by the resting fluorescence ( F 0 ), and the trace shown is for the islet in B. (D) Exocytic event (arrow) triggered by UV photolysis of NP-EGTA. Ω Profiles often persisted for tens of seconds. (E) Two examples of time courses of sequential exocytosis (blue and red) and an example of that of solitary exocytosis (black) triggered by photolysis of NP-EGTA. The traces were aligned at the onset of exocytosis. (F) Time courses of the decay of SRB fluorescence associated with exocytic events induced by 20 mM glucose (black and red) or by photolysis of NP-EGTA (green). The traces are averages of > 10 exocytic events, which were aligned at the peak of SRB fluorescence and normalized by the peak intensity. The data correspond to solitary events (black and green) or to the primary event of sequential exocytosis (red).

Techniques Used: Sulforhodamine B Assay, Fluorescence, Irradiation

6) Product Images from "PKA-dependent potentiation of glucose-stimulated insulin secretion by Epac activator 8-pCPT-2?-O-Me-cAMP-AM in human islets of Langerhans"

Article Title: PKA-dependent potentiation of glucose-stimulated insulin secretion by Epac activator 8-pCPT-2?-O-Me-cAMP-AM in human islets of Langerhans

Journal: American Journal of Physiology - Endocrinology and Metabolism

doi: 10.1152/ajpendo.00630.2009

8-pCPT-2′- O -Me-cAMP-AM facilitates Ca 2+ -induced Ca 2+ release (CICR) in human β-cells. A : Ca 2+ was uncaged by delivering UV excitation light (arrows) to a β-cell loaded with NP-EGTA and equilibrated in SES containing 5.6 mM glucose.

Figure Legend Snippet: 8-pCPT-2′- O -Me-cAMP-AM facilitates Ca 2+ -induced Ca 2+ release (CICR) in human β-cells. A : Ca 2+ was uncaged by delivering UV excitation light (arrows) to a β-cell loaded with NP-EGTA and equilibrated in SES containing 5.6 mM glucose.

Techniques Used:

other:

Article Title: Acid contact in the rodent pulmonary alveolus causes proinflammatory signaling by membrane pore formation
Article Snippet: 2′,7′-Bis(2-carboxyethyl)-5 (and 6)-carboxyfluorescein-AM (BCECF-AM), calcein red-orange AM, fura 2-AM, fluo 4-AM, o -nitrophenyl EGTA-AM (NP-EGTA-AM), 2′,7′-dichlorodihydrofluorescein diacetate (DCF), and N -(3-triethylammoniumpropyl)-4-[4-(dibutyl-amino)stytyl]pyridinium dibromide FM1-43 were from Molecular Probes.

Incubation:

Article Title: Altered properties of quantal neurotransmitter release at endplates of mice lacking P/Q-type Ca2+ channels
Article Snippet: .. Muscles were incubated in a Ca2+ -free saline solution in the presence of EGTA-AM or DM-BAPTA-AM (Molecular Probes) or dimethyl sulfoxide, as described ( ). .. Presynaptic labeling was obtained by incubation with 8 μM FM1–43 for 15 min in a modified physiological solution containing 45 mM KCl, followed by three washes (10 min each) in physiological saline.

Fluorescence:

Article Title: Exocytotic mechanism studied by truncated and zero layer mutants of the C-terminus of SNAP-25
Article Snippet: .. Chemicals used were: nitrophenyl-EGTA (Molecular Probes, Eugene, OR), fura-2 (Texas Fluorescence Labs, Austin, TX), furaptra (Molecular Probes, Eugene, OR), CaCl2 (Sigma, St Louis, MO) and ATP (Boehringer Mannheim, Germany). ..

Conditioned Place Preference:

Article Title: Astrocyte activation of presynaptic metabotropic glutamate receptors modulates hippocampal inhibitory synaptic transmission
Article Snippet: .. L(+)-2-amino-4-phosphonobutyric acid (L-AP4), (2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine (DCG IV), (RS)-α-cyclopropyl-4-phosphonophenylglycine (CPPG), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) were purchased from Tocris Cookson Inc. Acetoxymethyl (AM) esters and potassium salts of NP-EGTA and fluo-4, BAPTA AM (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate) and pluronic F-127 were from Molecular Probes Inc. ..