ethylene glycol tetraacetic acid Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher egta
    Filamin A is locally translated in injured axons. A , C , E , and G , Western blots of control (− Ax ) and axotomized (+ Ax ) sciatic nerves treated with vehicle or cycloheximide ( CHX , A ), <t>EGTA</t> ( C ), <t>Gö6976</t> ( E ), and H-89 ( G ). B , D , F , and H , quantifications of filamin A levels in A , C , E , and G . Data are mean ± S.E. n = 3; ***, p
    Egta, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4658 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/egta/product/Thermo Fisher
    Average 99 stars, based on 4658 article reviews
    Price from $9.99 to $1999.99
    egta - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore egta
    Cinnamycin facilitates annexin translocation across membranes in liposomes but not galectin-3. (A) Cinnamycin induces lipid movement in liposomes. Left, schematic of the lipid quenching assay. Right, LUVs (PC:PE 1:1, and a trace of <t>NBD-PE)</t> were pre-incubated with cinnamycin (10 µM) or DMSO for 30 min and changes in NBD fluorescence during the experiment time were recorded upon the addition of dithionite and Triton X-100. (B) Cinnamycin increases the <t>EGTA-resistant</t> membrane-bound fraction of annexin A5. PC:PE liposomes (MLVs) in CaCl 2 -containing buffer were incubated with annexin A5. Cinnamycin or DMSO was added for a further 40 min incubation at 37°C. Some of the samples were also treated with EGTA before all samples were centrifuged. Liposome pellets were mixed with boiling SDS sample buffer, separated by SDS-PAGE and analysed by western blotted with anti-annexin A5 antibodies for the detection of the membrane-bound fraction of annexin A5. Results are mean±s.d. comparing lane 3 vs. lane 4; n =3 independent experiments, * P
    Egta, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 21948 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/egta/product/Millipore
    Average 99 stars, based on 21948 article reviews
    Price from $9.99 to $1999.99
    egta - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore ethylene glycol tetraacetic acid egta
    Roles of ROS and TRPM8 in the increased intracellular Ca 2+ level induced by M-CSE and Non-M-CSE in HBECs . Intracellular Ca 2+ levels were measured by Fluo-8 fluorescent probe assay. (A) Cells were exposed to medium alone (control), 1% Non-M-CSE, or 1% M-CSE for 1, 2, 5, 10, and 30 min. (B) Representative images of fluorescence-positive cells at 5 min after exposure. (C) Cells were pretreated with N-acetyl-cysteine (NAC), AMTB, or ethylene glycol <t>tetraacetic</t> acid <t>(EGTA)</t> for 1 h and then exposed to medium alone, 1% Non-M-CSE, or 1% M-CSE for 5 min. (D) Cells were transfected with control plasmid or TRPM8 Double Nickase plasmid (KO) and then exposed to medium alone, 1% Non-M-CSE, or 1% M-CSE for 5 min. Data from each group are means ± SEM from four independent experiments. * p
    Ethylene Glycol Tetraacetic Acid Egta, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 799 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ethylene glycol tetraacetic acid egta/product/Millipore
    Average 99 stars, based on 799 article reviews
    Price from $9.99 to $1999.99
    ethylene glycol tetraacetic acid egta - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    egta  (Tocris)
    99
    Tocris egta
    Protective effects of PMC-12 on cellular Ca 2+ -related ROS production in HT22 cells. Cells were pretreated with 10 μg/ml of PMC-12 for 24 h, followed by exposure to 5 mM glutamate for 24 h. Cells were treated with the specific Ca 2+ inhibitors, 1.5 mM <t>EGTA</t> or 10 μM <t>BAPTA-AM,</t> for 30 min before addition of PMC-12 or glutamate. ROS production was measured using a fluorescence plate reader and mean fluorescence intensity was expressed as the mean ± SEM of three independent experiments. ### P
    Egta, supplied by Tocris, used in various techniques. Bioz Stars score: 99/100, based on 275 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/egta/product/Tocris
    Average 99 stars, based on 275 article reviews
    Price from $9.99 to $1999.99
    egta - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    95
    Thermo Fisher egta am
    (A) Effect of calcium chelators and K channel antagonist, ChTX on fEPSP during OGD. 1μM <t>BAPTA-AM</t> increases the amount of evoked neurotransmission remaining after 2 min (n = 7), 4 min (n = 6) and 6 min (n = 5) of ischemia relative to control (n = 7, 5, 5, respectively). Similarly, the amplitude of fEPSPs remaining after 2 min, 4 min, 6 min of OGD (n = 6) is increased after administration of 10 nM ChTX. Combining ChTX and BAPTA-AM led to almost no change in fEPSP amplitude up to 6 min of OGD (n = 6). ( B) Effect of calcium chelators, and BK channel antagonist on recovery of fEPSP after OGD. BAPTA-AM (1 μM) decreases recovery time from 2 min (n = 7), 4 min (n = 6) and 6 min (n = 3) of in vitro OGD compared to control (n = 7, 5, 5, respectively). <t>EGTA-AM</t> (50μM) shows similar effects to BAPTA-AM (1μM) by decreasing time needed for electrophysiological recovery after 4 min of OGD (n = 6) but not after 6 min (n = 7). ChTX (10 nM) did not significantly decrease recovery time after 6 min of ischemia (n = 4) but a combination of BAPTA-AM (1 μM) and ChTX(10 nM) significantly decreased recovery time after 6 min of OGD (n = 6). ( C) BAPTA-AM and BAPTA-AM + ChTX promote increased tissue resistance to a long ischemic episode. BAPTA-AM (1 μM) increases the amount of fEPSP remaining after 8 min of OGD and leads to full recovery after 40 min of reperfusion post ischemia (n = 3) when control tissue has surpassed the point of functional recovery (n = 4). A combination of BAPTA-AM (1 μM) and ChTX (10 nM) leads to almost no change in fEPSP amplitude after prolonged OGD and leads to almost full recovery after 40 min of reperfusion (n = 5). Data plotted as mean ± SE. * p
    Egta Am, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 264 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/egta am/product/Thermo Fisher
    Average 95 stars, based on 264 article reviews
    Price from $9.99 to $1999.99
    egta am - by Bioz Stars, 2020-09
    95/100 stars
      Buy from Supplier

    92
    AnaSpec egta am
    Stimulation of CREB-dependent transcription by forskolin requires an increase in [Ca 2+ ] within a microdomain. One day after transfecting with 4x CRE reporter plasmid, cultures were preincubated with <t>Bapta-AM</t> or <t>EGTA-AM</t> (10 μM) for 1 hr and then
    Egta Am, supplied by AnaSpec, used in various techniques. Bioz Stars score: 92/100, based on 99 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/egta am/product/AnaSpec
    Average 92 stars, based on 99 article reviews
    Price from $9.99 to $1999.99
    egta am - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    92
    Boston BioProducts egta
    Stimulation of CREB-dependent transcription by forskolin requires an increase in [Ca 2+ ] within a microdomain. One day after transfecting with 4x CRE reporter plasmid, cultures were preincubated with <t>Bapta-AM</t> or <t>EGTA-AM</t> (10 μM) for 1 hr and then
    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
    https://www.bioz.com/result/egta/product/Boston BioProducts
    Average 92 stars, based on 177 article reviews
    Price from $9.99 to $1999.99
    egta - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    93
    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.
    Egta, supplied by Dojindo Labs, used in various techniques. Bioz Stars score: 93/100, based on 106 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/egta/product/Dojindo Labs
    Average 93 stars, based on 106 article reviews
    Price from $9.99 to $1999.99
    egta - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    93
    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: 93/100, based on 299 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/egta/product/Fisher Scientific
    Average 93 stars, based on 299 article reviews
    Price from $9.99 to $1999.99
    egta - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    92
    Merck & Co egta
    Effect of Ca 2+ chelation and Ca 2+ channel inhibitors on HMR 3647 uptake. (A) PMNs were incubated for 5 to 60 min in the presence of 1 mM <t>EGTA,</t> 5 mM Ni 2+ , 125 μM verapamil (VPL), or control <t>HBSS.</t> Results are means ± SEM for four to six experiments. ∗, P
    Egta, supplied by Merck & Co, used in various techniques. Bioz Stars score: 92/100, based on 233 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/egta/product/Merck & Co
    Average 92 stars, based on 233 article reviews
    Price from $9.99 to $1999.99
    egta - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    99
    Millipore ethylene glycol bis beta aminoethyl ether n n n n tetraacetic acid tetrasodium salt
    Origin and nature of n -Hex- and DCM-EOM-induced [Ca 2+ ] i responses. Cells were incubated in a buffer with or without Ca 2+ and ethylene glycol-bis(β-aminoethyl ether)- N , N , N ′, N <t>′-tetraacetic</t> acid <t>(EGTA),</t> 30 min before exposure. Three min after measurements were started, the cells were exposed to n -Hex- ( A ) or DCM-EOM ( B ) at concentrations corresponding to 5 μg/mL of the original DEPs or vehicle control (DMSO). [Ca 2+ ] i level measured by normalized ratio of the Fura2-AM probe during exposure is presented as a graph and AUC from 1.0 at the Y -axis and 0–45 min, as mean and mean ± SEM ( n = 3), respectively. * Significantly different from calcium free exposure with EGTA ( p
    Ethylene Glycol Bis Beta Aminoethyl Ether N N N N Tetraacetic Acid Tetrasodium Salt, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ethylene glycol bis beta aminoethyl ether n n n n tetraacetic acid tetrasodium salt/product/Millipore
    Average 99 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    ethylene glycol bis beta aminoethyl ether n n n n tetraacetic acid tetrasodium salt - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    84
    Thermo Fisher egta tetra acetoxymethyl ester egta am
    Origin and nature of n -Hex- and DCM-EOM-induced [Ca 2+ ] i responses. Cells were incubated in a buffer with or without Ca 2+ and ethylene glycol-bis(β-aminoethyl ether)- N , N , N ′, N <t>′-tetraacetic</t> acid <t>(EGTA),</t> 30 min before exposure. Three min after measurements were started, the cells were exposed to n -Hex- ( A ) or DCM-EOM ( B ) at concentrations corresponding to 5 μg/mL of the original DEPs or vehicle control (DMSO). [Ca 2+ ] i level measured by normalized ratio of the Fura2-AM probe during exposure is presented as a graph and AUC from 1.0 at the Y -axis and 0–45 min, as mean and mean ± SEM ( n = 3), respectively. * Significantly different from calcium free exposure with EGTA ( p
    Egta Tetra Acetoxymethyl Ester Egta Am, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/egta tetra acetoxymethyl ester egta am/product/Thermo Fisher
    Average 84 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    egta tetra acetoxymethyl ester egta am - by Bioz Stars, 2020-09
    84/100 stars
      Buy from Supplier

    92
    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).
    Egta, supplied by Carl Roth GmbH, used in various techniques. Bioz Stars score: 92/100, based on 120 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/egta/product/Carl Roth GmbH
    Average 92 stars, based on 120 article reviews
    Price from $9.99 to $1999.99
    egta - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    99
    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).
    Egta Ethylene Glycol Bis 2 Aminoethylether N, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/egta ethylene glycol bis 2 aminoethylether n/product/Millipore
    Average 99 stars, based on 71 article reviews
    Price from $9.99 to $1999.99
    egta ethylene glycol bis 2 aminoethylether n - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    93
    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
    Egta, supplied by Amresco, used in various techniques. Bioz Stars score: 93/100, based on 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/egta/product/Amresco
    Average 93 stars, based on 83 article reviews
    Price from $9.99 to $1999.99
    egta - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    88
    Thermo Fisher trypsin 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
    Trypsin Egta, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/trypsin egta/product/Thermo Fisher
    Average 88 stars, based on 40 article reviews
    Price from $9.99 to $1999.99
    trypsin egta - by Bioz Stars, 2020-09
    88/100 stars
      Buy from Supplier

    Image Search Results


    Filamin A is locally translated in injured axons. A , C , E , and G , Western blots of control (− Ax ) and axotomized (+ Ax ) sciatic nerves treated with vehicle or cycloheximide ( CHX , A ), EGTA ( C ), Gö6976 ( E ), and H-89 ( G ). B , D , F , and H , quantifications of filamin A levels in A , C , E , and G . Data are mean ± S.E. n = 3; ***, p

    Journal: The Journal of Biological Chemistry

    Article Title: Filamin A Is Required in Injured Axons for HDAC5 Activity and Axon Regeneration *

    doi: 10.1074/jbc.M115.638445

    Figure Lengend Snippet: Filamin A is locally translated in injured axons. A , C , E , and G , Western blots of control (− Ax ) and axotomized (+ Ax ) sciatic nerves treated with vehicle or cycloheximide ( CHX , A ), EGTA ( C ), Gö6976 ( E ), and H-89 ( G ). B , D , F , and H , quantifications of filamin A levels in A , C , E , and G . Data are mean ± S.E. n = 3; ***, p

    Article Snippet: For sciatic nerve delivery of chemical compounds, the chemicals cycloheximide (1 mg/kg, Sigma), EGTA (10 m m , Life Technologies), Gö6976 (1 mg/kg, Tocris), and H-89 (1 mg/kg, Tocris) were dissolved in dimethyl sulfoxide.

    Techniques: Western Blot

    Cinnamycin facilitates annexin translocation across membranes in liposomes but not galectin-3. (A) Cinnamycin induces lipid movement in liposomes. Left, schematic of the lipid quenching assay. Right, LUVs (PC:PE 1:1, and a trace of NBD-PE) were pre-incubated with cinnamycin (10 µM) or DMSO for 30 min and changes in NBD fluorescence during the experiment time were recorded upon the addition of dithionite and Triton X-100. (B) Cinnamycin increases the EGTA-resistant membrane-bound fraction of annexin A5. PC:PE liposomes (MLVs) in CaCl 2 -containing buffer were incubated with annexin A5. Cinnamycin or DMSO was added for a further 40 min incubation at 37°C. Some of the samples were also treated with EGTA before all samples were centrifuged. Liposome pellets were mixed with boiling SDS sample buffer, separated by SDS-PAGE and analysed by western blotted with anti-annexin A5 antibodies for the detection of the membrane-bound fraction of annexin A5. Results are mean±s.d. comparing lane 3 vs. lane 4; n =3 independent experiments, * P

    Journal: Journal of Cell Science

    Article Title: Transbilayer phospholipid movement facilitates the translocation of annexin across membranes

    doi: 10.1242/jcs.217034

    Figure Lengend Snippet: Cinnamycin facilitates annexin translocation across membranes in liposomes but not galectin-3. (A) Cinnamycin induces lipid movement in liposomes. Left, schematic of the lipid quenching assay. Right, LUVs (PC:PE 1:1, and a trace of NBD-PE) were pre-incubated with cinnamycin (10 µM) or DMSO for 30 min and changes in NBD fluorescence during the experiment time were recorded upon the addition of dithionite and Triton X-100. (B) Cinnamycin increases the EGTA-resistant membrane-bound fraction of annexin A5. PC:PE liposomes (MLVs) in CaCl 2 -containing buffer were incubated with annexin A5. Cinnamycin or DMSO was added for a further 40 min incubation at 37°C. Some of the samples were also treated with EGTA before all samples were centrifuged. Liposome pellets were mixed with boiling SDS sample buffer, separated by SDS-PAGE and analysed by western blotted with anti-annexin A5 antibodies for the detection of the membrane-bound fraction of annexin A5. Results are mean±s.d. comparing lane 3 vs. lane 4; n =3 independent experiments, * P

    Article Snippet: Reagents Reagents used were: cinnamycin (Santa Cruz Biotechnology; sc-391464), mastoparan X (Alfa Aesar; J61173), recombinant annexin-A5–FITC (Abcam; ab14085), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, Sigma-Aldrich; 54008), 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC, Sigma-Aldrich; P3017), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE, Avanti Polar Lipids, 810145) and proteinase K (Molecular Biology; BP1700-100), EGTA (Sigma-Aldrich; E3889), sodium dithionite (Sigma-Aldrich; 71699), versene solution containing ethylenediaminetetraacetic acid (EDTA) (Gibco; 15040-033), propidium iodide solution (Biolegend; 421301), QuickExtract DNA extraction solution (Epicenter; QE0905T), Herculase II fusion DNA polymerase (Agilent; 600675), annexin-V–FITC and annexin-V–Cy5 Apoptosis Staining/Detection Kit (ab14085, ab14150), annexin-V conjugated to Alexa Fluor 647 (Biolegend; 640912) and ionomycin (Cayman Chemical Company; 10004974), recombinant galectin-3 (Biolegend; 599706) and recombinant anneaxin A5 (Novus NBP1-30265).

    Techniques: Translocation Assay, Incubation, Fluorescence, SDS Page, Western Blot

    Roles of ROS and TRPM8 in the increased intracellular Ca 2+ level induced by M-CSE and Non-M-CSE in HBECs . Intracellular Ca 2+ levels were measured by Fluo-8 fluorescent probe assay. (A) Cells were exposed to medium alone (control), 1% Non-M-CSE, or 1% M-CSE for 1, 2, 5, 10, and 30 min. (B) Representative images of fluorescence-positive cells at 5 min after exposure. (C) Cells were pretreated with N-acetyl-cysteine (NAC), AMTB, or ethylene glycol tetraacetic acid (EGTA) for 1 h and then exposed to medium alone, 1% Non-M-CSE, or 1% M-CSE for 5 min. (D) Cells were transfected with control plasmid or TRPM8 Double Nickase plasmid (KO) and then exposed to medium alone, 1% Non-M-CSE, or 1% M-CSE for 5 min. Data from each group are means ± SEM from four independent experiments. * p

    Journal: Frontiers in Physiology

    Article Title: Inflammatory Effects of Menthol vs. Non-menthol Cigarette Smoke Extract on Human Lung Epithelial Cells: A Double-Hit on TRPM8 by Reactive Oxygen Species and Menthol

    doi: 10.3389/fphys.2017.00263

    Figure Lengend Snippet: Roles of ROS and TRPM8 in the increased intracellular Ca 2+ level induced by M-CSE and Non-M-CSE in HBECs . Intracellular Ca 2+ levels were measured by Fluo-8 fluorescent probe assay. (A) Cells were exposed to medium alone (control), 1% Non-M-CSE, or 1% M-CSE for 1, 2, 5, 10, and 30 min. (B) Representative images of fluorescence-positive cells at 5 min after exposure. (C) Cells were pretreated with N-acetyl-cysteine (NAC), AMTB, or ethylene glycol tetraacetic acid (EGTA) for 1 h and then exposed to medium alone, 1% Non-M-CSE, or 1% M-CSE for 5 min. (D) Cells were transfected with control plasmid or TRPM8 Double Nickase plasmid (KO) and then exposed to medium alone, 1% Non-M-CSE, or 1% M-CSE for 5 min. Data from each group are means ± SEM from four independent experiments. * p

    Article Snippet: Mouse Ab against α-tubulin, mouse Ab against FLAG, ethylene glycol tetraacetic acid (EGTA), N-acetyl-cysteine (NAC), apocynin, and L-menthol (purity > 99%, FCC) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Fluorescence, Transfection, Plasmid Preparation

    Ca 2+ influx via TRPM2 was required for HG-induced NLRP3 inflammasome activation in U937 cells. ( A ) Fluorescence images showing fluo-4-loaded cells taken at indicated timepoints, and representative flow cytometric plot and graph showing relative median fluorescence intensity (MFI) of intracellular Ca 2+ concentration by Fluo-4 staining under low glucose (LG; 5.5 mM glucose;) or high glucose (HG; 30 mM glucose) (n = 5). ( B ) Relative changes in [Ca 2+ ] i , evoked by H 2 O 2 (1 mM) over the time course. The cells were treated in the presence of 2-aminoethoxydiphenyl borate (2-APB; 100 μM), or with pre-treatment of 3-aminobenzamide (3-AB; 5 mM) or 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ; 100 μM), or GAPDH- (GA si) or TRPM2-siRNAs (TRPM2 si) under HG (n = 4). ( C ) Representative immunoblots for pro-IL-1β, IL-1β p17, pro-caspase-1, cleaved caspase-1 (p20), and GAPDH in the presence of EGTA-AM (0.5, 1, 5 mM) under HG (n = 4). ( D ) Immunofluorescence images showing the location of caspase-1 and ASC in fixed cells using confocal microscopy in the presence of GAPDH- or TRPM2-siRNAs, or EGTA-AM (5 mM) under HG. The percentage of co-localization of caspase-1 with ASC was calculated as the average volume of the overlapping areas (n = 4). Data were shown as mean ± S.E.M. ( A ) *P

    Journal: Scientific Reports

    Article Title: TRPM2 regulates TXNIP-mediated NLRP3 inflammasome activation via interaction with p47 phox under high glucose in human monocytic cells

    doi: 10.1038/srep35016

    Figure Lengend Snippet: Ca 2+ influx via TRPM2 was required for HG-induced NLRP3 inflammasome activation in U937 cells. ( A ) Fluorescence images showing fluo-4-loaded cells taken at indicated timepoints, and representative flow cytometric plot and graph showing relative median fluorescence intensity (MFI) of intracellular Ca 2+ concentration by Fluo-4 staining under low glucose (LG; 5.5 mM glucose;) or high glucose (HG; 30 mM glucose) (n = 5). ( B ) Relative changes in [Ca 2+ ] i , evoked by H 2 O 2 (1 mM) over the time course. The cells were treated in the presence of 2-aminoethoxydiphenyl borate (2-APB; 100 μM), or with pre-treatment of 3-aminobenzamide (3-AB; 5 mM) or 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ; 100 μM), or GAPDH- (GA si) or TRPM2-siRNAs (TRPM2 si) under HG (n = 4). ( C ) Representative immunoblots for pro-IL-1β, IL-1β p17, pro-caspase-1, cleaved caspase-1 (p20), and GAPDH in the presence of EGTA-AM (0.5, 1, 5 mM) under HG (n = 4). ( D ) Immunofluorescence images showing the location of caspase-1 and ASC in fixed cells using confocal microscopy in the presence of GAPDH- or TRPM2-siRNAs, or EGTA-AM (5 mM) under HG. The percentage of co-localization of caspase-1 with ASC was calculated as the average volume of the overlapping areas (n = 4). Data were shown as mean ± S.E.M. ( A ) *P

    Article Snippet: Reagents and chemicals 2-aminoethoxydiphenyl borate (2-APB), 3-aminobenzamide (3-AB), adenosine 5′-diphosphoribose (ADPR), adenosine monophosphate (AMP), ethylene glycol tetra acetic acid (EGTA), hydrogen peroxide solution (H2 O2 ), D-mannitol, N-acetyl-L-cysteine (NAC) were purchased from Sigma-Aldrich, US.

    Techniques: Activation Assay, Fluorescence, Flow Cytometry, Concentration Assay, Staining, Western Blot, Immunofluorescence, Confocal Microscopy

    Protective effects of PMC-12 on cellular Ca 2+ -related ROS production in HT22 cells. Cells were pretreated with 10 μg/ml of PMC-12 for 24 h, followed by exposure to 5 mM glutamate for 24 h. Cells were treated with the specific Ca 2+ inhibitors, 1.5 mM EGTA or 10 μM BAPTA-AM, for 30 min before addition of PMC-12 or glutamate. ROS production was measured using a fluorescence plate reader and mean fluorescence intensity was expressed as the mean ± SEM of three independent experiments. ### P

    Journal: BMC Complementary and Alternative Medicine

    Article Title: Neuroprotection and spatial memory enhancement of four herbal mixture extract in HT22 hippocampal cells and a mouse model of focal cerebral ischemia

    doi: 10.1186/s12906-015-0741-1

    Figure Lengend Snippet: Protective effects of PMC-12 on cellular Ca 2+ -related ROS production in HT22 cells. Cells were pretreated with 10 μg/ml of PMC-12 for 24 h, followed by exposure to 5 mM glutamate for 24 h. Cells were treated with the specific Ca 2+ inhibitors, 1.5 mM EGTA or 10 μM BAPTA-AM, for 30 min before addition of PMC-12 or glutamate. ROS production was measured using a fluorescence plate reader and mean fluorescence intensity was expressed as the mean ± SEM of three independent experiments. ### P

    Article Snippet: BAPTA-AM and EGTA were purchased from Tocris Bioscience (Ellisville, MO, USA).

    Techniques: Fluorescence

    (A) Effect of calcium chelators and K channel antagonist, ChTX on fEPSP during OGD. 1μM BAPTA-AM increases the amount of evoked neurotransmission remaining after 2 min (n = 7), 4 min (n = 6) and 6 min (n = 5) of ischemia relative to control (n = 7, 5, 5, respectively). Similarly, the amplitude of fEPSPs remaining after 2 min, 4 min, 6 min of OGD (n = 6) is increased after administration of 10 nM ChTX. Combining ChTX and BAPTA-AM led to almost no change in fEPSP amplitude up to 6 min of OGD (n = 6). ( B) Effect of calcium chelators, and BK channel antagonist on recovery of fEPSP after OGD. BAPTA-AM (1 μM) decreases recovery time from 2 min (n = 7), 4 min (n = 6) and 6 min (n = 3) of in vitro OGD compared to control (n = 7, 5, 5, respectively). EGTA-AM (50μM) shows similar effects to BAPTA-AM (1μM) by decreasing time needed for electrophysiological recovery after 4 min of OGD (n = 6) but not after 6 min (n = 7). ChTX (10 nM) did not significantly decrease recovery time after 6 min of ischemia (n = 4) but a combination of BAPTA-AM (1 μM) and ChTX(10 nM) significantly decreased recovery time after 6 min of OGD (n = 6). ( C) BAPTA-AM and BAPTA-AM + ChTX promote increased tissue resistance to a long ischemic episode. BAPTA-AM (1 μM) increases the amount of fEPSP remaining after 8 min of OGD and leads to full recovery after 40 min of reperfusion post ischemia (n = 3) when control tissue has surpassed the point of functional recovery (n = 4). A combination of BAPTA-AM (1 μM) and ChTX (10 nM) leads to almost no change in fEPSP amplitude after prolonged OGD and leads to almost full recovery after 40 min of reperfusion (n = 5). Data plotted as mean ± SE. * p

    Journal: PLoS ONE

    Article Title: Raised Intracellular Calcium Contributes to Ischemia-Induced Depression of Evoked Synaptic Transmission

    doi: 10.1371/journal.pone.0148110

    Figure Lengend Snippet: (A) Effect of calcium chelators and K channel antagonist, ChTX on fEPSP during OGD. 1μM BAPTA-AM increases the amount of evoked neurotransmission remaining after 2 min (n = 7), 4 min (n = 6) and 6 min (n = 5) of ischemia relative to control (n = 7, 5, 5, respectively). Similarly, the amplitude of fEPSPs remaining after 2 min, 4 min, 6 min of OGD (n = 6) is increased after administration of 10 nM ChTX. Combining ChTX and BAPTA-AM led to almost no change in fEPSP amplitude up to 6 min of OGD (n = 6). ( B) Effect of calcium chelators, and BK channel antagonist on recovery of fEPSP after OGD. BAPTA-AM (1 μM) decreases recovery time from 2 min (n = 7), 4 min (n = 6) and 6 min (n = 3) of in vitro OGD compared to control (n = 7, 5, 5, respectively). EGTA-AM (50μM) shows similar effects to BAPTA-AM (1μM) by decreasing time needed for electrophysiological recovery after 4 min of OGD (n = 6) but not after 6 min (n = 7). ChTX (10 nM) did not significantly decrease recovery time after 6 min of ischemia (n = 4) but a combination of BAPTA-AM (1 μM) and ChTX(10 nM) significantly decreased recovery time after 6 min of OGD (n = 6). ( C) BAPTA-AM and BAPTA-AM + ChTX promote increased tissue resistance to a long ischemic episode. BAPTA-AM (1 μM) increases the amount of fEPSP remaining after 8 min of OGD and leads to full recovery after 40 min of reperfusion post ischemia (n = 3) when control tissue has surpassed the point of functional recovery (n = 4). A combination of BAPTA-AM (1 μM) and ChTX (10 nM) leads to almost no change in fEPSP amplitude after prolonged OGD and leads to almost full recovery after 40 min of reperfusion (n = 5). Data plotted as mean ± SE. * p

    Article Snippet: Bis-(o -aminophenoxy)ethane-N ,N ,N ′,N ′-tetraacetic acid acetoxymethyl ester (BAPTA-AM) and EGTA-AM (Molecular Probes, Eugene, OR) were initially dissolved in DMSO, and then diluted to their final concentrations in the ACSF.

    Techniques: In Vitro, Functional Assay

    Cell-permeant calcium chelators reduce ischemia-induced depression of fEPSP amplitudes. (A) Time course of the depression and subsequent recovery of fEPSP amplitudes in drug (left) and control (right) condition. Left Calcium chelator data. 4min OGD in the presence EGTA-AM (grey triangle, n = 6) or BAPTA-AM (black circle, n = 6) leads to a smaller depression of fEPSP amplitudes relative to control, along with faster recovery of the response (approximately 5–6 min). Right Control data. 4min of OGD produces a large depression of fEPSP amplitude, which then takes approximately 9 min to recover (n = 5). (B) Amount of fEPSP amplitude remaining after oxygen-glucose deprivation. 1 μM BAPTA-AM increases the amount of evoked neurotransmission remaining after 2 min (n = 7), 4 min (n = 6) and 6 min (n = 5) of ischemia relative to control (n = 7, 5, 5, respectively). 50 μM EGTA-AM (n = 6) shows similar effects to BAPTA-AM (1 μM) at 4 min of OGD, with both chelators increasing the fEPSP amplitude remaining after OGD. At 6 min however, EGTA-AM does not significantly reduce OGD-induced depression of fEPSP amplitude relative to control (n = 6) Data plotted as mean ± SE. * p

    Journal: PLoS ONE

    Article Title: Raised Intracellular Calcium Contributes to Ischemia-Induced Depression of Evoked Synaptic Transmission

    doi: 10.1371/journal.pone.0148110

    Figure Lengend Snippet: Cell-permeant calcium chelators reduce ischemia-induced depression of fEPSP amplitudes. (A) Time course of the depression and subsequent recovery of fEPSP amplitudes in drug (left) and control (right) condition. Left Calcium chelator data. 4min OGD in the presence EGTA-AM (grey triangle, n = 6) or BAPTA-AM (black circle, n = 6) leads to a smaller depression of fEPSP amplitudes relative to control, along with faster recovery of the response (approximately 5–6 min). Right Control data. 4min of OGD produces a large depression of fEPSP amplitude, which then takes approximately 9 min to recover (n = 5). (B) Amount of fEPSP amplitude remaining after oxygen-glucose deprivation. 1 μM BAPTA-AM increases the amount of evoked neurotransmission remaining after 2 min (n = 7), 4 min (n = 6) and 6 min (n = 5) of ischemia relative to control (n = 7, 5, 5, respectively). 50 μM EGTA-AM (n = 6) shows similar effects to BAPTA-AM (1 μM) at 4 min of OGD, with both chelators increasing the fEPSP amplitude remaining after OGD. At 6 min however, EGTA-AM does not significantly reduce OGD-induced depression of fEPSP amplitude relative to control (n = 6) Data plotted as mean ± SE. * p

    Article Snippet: Bis-(o -aminophenoxy)ethane-N ,N ,N ′,N ′-tetraacetic acid acetoxymethyl ester (BAPTA-AM) and EGTA-AM (Molecular Probes, Eugene, OR) were initially dissolved in DMSO, and then diluted to their final concentrations in the ACSF.

    Techniques:

    Neuronal loading of NP-EGTA in NP-EGTA-AM-loaded slices is insignificant. (A1) A pyramidal neuron was loaded with potassium salts of NP-EGTA (2 mM, pre-loaded with 2 mM Ca 2+ ) and fluo-4 (0.1 mM) through conventional whole-cell recording. Uncaging NP-EGTA induced Ca 2+ elevation as shown by the change in fluo-4 fluorescence. Scale bar, 15 μm. (A2) Time-course of ΔF/F 0 of the experiment shown in A1. (A3) Uncaging NP-EGTA induced Ca 2+ -activated K + current in the pyramidal neuron. (B) Perforated whole-cell recordings from a pyramidal neuron in NP-EGTA-AM-loaded slices. UV flashing to the neuron did not activate Ca 2+ -activated K + currents.

    Journal: Neuron glia biology

    Article Title: Astrocyte activation of presynaptic metabotropic glutamate receptors modulates hippocampal inhibitory synaptic transmission

    doi: 10.1017/S1740925X05000190

    Figure Lengend Snippet: Neuronal loading of NP-EGTA in NP-EGTA-AM-loaded slices is insignificant. (A1) A pyramidal neuron was loaded with potassium salts of NP-EGTA (2 mM, pre-loaded with 2 mM Ca 2+ ) and fluo-4 (0.1 mM) through conventional whole-cell recording. Uncaging NP-EGTA induced Ca 2+ elevation as shown by the change in fluo-4 fluorescence. Scale bar, 15 μm. (A2) Time-course of ΔF/F 0 of the experiment shown in A1. (A3) Uncaging NP-EGTA induced Ca 2+ -activated K + current in the pyramidal neuron. (B) Perforated whole-cell recordings from a pyramidal neuron in NP-EGTA-AM-loaded slices. UV flashing to the neuron did not activate Ca 2+ -activated K + currents.

    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.

    Techniques: Fluorescence

    Depression of eIPSCs in interneurons by Ca 2+ uncaging in astrocytes. (A) Images from an astrocyte before, during and after stimulation with a train of twelve UV laser pulses (0.1 Hz) to uncage NP-EGTA. Scale bar, 10 μm. (B) Upper : Ca 2+ uncaging produces a stepwise increase in ΔF/F 0 of the astrocyte in (A) and a reversible decrease in the mean amplitude of eIPSCs (averaged from 24–36 traces) in an interneuron ( middle ). The amplitudes of eIPSCs over the course of the experiment ( lower ). (C) Normalized changes in the amplitude of eIPSCs by uncaging. Responder shows the average of nine cells that responded to uncaging by decreasing the amplitude of eIPSCs. Non-Responder shows the average of eight cells that did not respond to uncaging. Total shows the average of all 17 cells. Preloading the slices with BAPTA-AM (10 μM), a calcium chelator, prevents the depression of eIPSCs by uncaging ( n = 8). No NP-EGTA indicates slices loaded with fluo-4 alone ( n = 9). * P

    Journal: Neuron glia biology

    Article Title: Astrocyte activation of presynaptic metabotropic glutamate receptors modulates hippocampal inhibitory synaptic transmission

    doi: 10.1017/S1740925X05000190

    Figure Lengend Snippet: Depression of eIPSCs in interneurons by Ca 2+ uncaging in astrocytes. (A) Images from an astrocyte before, during and after stimulation with a train of twelve UV laser pulses (0.1 Hz) to uncage NP-EGTA. Scale bar, 10 μm. (B) Upper : Ca 2+ uncaging produces a stepwise increase in ΔF/F 0 of the astrocyte in (A) and a reversible decrease in the mean amplitude of eIPSCs (averaged from 24–36 traces) in an interneuron ( middle ). The amplitudes of eIPSCs over the course of the experiment ( lower ). (C) Normalized changes in the amplitude of eIPSCs by uncaging. Responder shows the average of nine cells that responded to uncaging by decreasing the amplitude of eIPSCs. Non-Responder shows the average of eight cells that did not respond to uncaging. Total shows the average of all 17 cells. Preloading the slices with BAPTA-AM (10 μM), a calcium chelator, prevents the depression of eIPSCs by uncaging ( n = 8). No NP-EGTA indicates slices loaded with fluo-4 alone ( n = 9). * P

    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.

    Techniques:

    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

    Journal: The EMBO Journal

    Article Title: Intracellular calcium changes trigger connexin 32 hemichannel opening

    doi: 10.1038/sj.emboj.7600908

    Figure Lengend 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

    Article Snippet: Fluo-3 acetoxymethyl ester (fluo-3-AM), fura-2-AM, NP-EGTA-AM, ethylenedioxybis( o -phenylenenitrilo)tetraacetic acid acetoxymethyl ester (BAPTA-AM), 4-bromo-A23187 (A23187), 6-carboxyfluorescein (6-CF), dextran fluorescein conjugate (MW 10 kDa) and PI were obtained from Molecular Probes (Leiden, The Netherlands).

    Techniques: Incubation, Fluorescence, Concentration Assay

    Stimulation of CREB-dependent transcription by forskolin requires an increase in [Ca 2+ ] within a microdomain. One day after transfecting with 4x CRE reporter plasmid, cultures were preincubated with Bapta-AM or EGTA-AM (10 μM) for 1 hr and then

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Autocrine activation of neuronal NMDA receptors by aspartate mediates dopamine- and cAMP-induced CREB-dependent gene transcription

    doi: 10.1523/JNEUROSCI.1166-09.2009

    Figure Lengend Snippet: Stimulation of CREB-dependent transcription by forskolin requires an increase in [Ca 2+ ] within a microdomain. One day after transfecting with 4x CRE reporter plasmid, cultures were preincubated with Bapta-AM or EGTA-AM (10 μM) for 1 hr and then

    Article Snippet: Antagonists were added 30 minutes before stimulation except for Bapta-AM and EGTA-AM which were added 60 before stimulation.

    Techniques: Plasmid Preparation

    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

    Effect of Ca 2+ chelation and Ca 2+ channel inhibitors on HMR 3647 uptake. (A) PMNs were incubated for 5 to 60 min in the presence of 1 mM EGTA, 5 mM Ni 2+ , 125 μM verapamil (VPL), or control HBSS. Results are means ± SEM for four to six experiments. ∗, P

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Interactions between HMR 3647, a New Ketolide, and Human Polymorphonuclear Neutrophils

    doi:

    Figure Lengend Snippet: Effect of Ca 2+ chelation and Ca 2+ channel inhibitors on HMR 3647 uptake. (A) PMNs were incubated for 5 to 60 min in the presence of 1 mM EGTA, 5 mM Ni 2+ , 125 μM verapamil (VPL), or control HBSS. Results are means ± SEM for four to six experiments. ∗, P

    Article Snippet: The uptake kinetics of macrolides was assessed first in Ca2+ -depleted HBSS (Gibco), supplemented with 1 mM EGTA (Merck), 1 mM magnesium chloride (Merck), and 4.2 mM sodium bicarbonate (NaHCO3 ) (Diagnostic Pasteur).

    Techniques: Incubation

    Origin and nature of n -Hex- and DCM-EOM-induced [Ca 2+ ] i responses. Cells were incubated in a buffer with or without Ca 2+ and ethylene glycol-bis(β-aminoethyl ether)- N , N , N ′, N ′-tetraacetic acid (EGTA), 30 min before exposure. Three min after measurements were started, the cells were exposed to n -Hex- ( A ) or DCM-EOM ( B ) at concentrations corresponding to 5 μg/mL of the original DEPs or vehicle control (DMSO). [Ca 2+ ] i level measured by normalized ratio of the Fura2-AM probe during exposure is presented as a graph and AUC from 1.0 at the Y -axis and 0–45 min, as mean and mean ± SEM ( n = 3), respectively. * Significantly different from calcium free exposure with EGTA ( p

    Journal: International Journal of Molecular Sciences

    Article Title: Lipophilic Chemicals from Diesel Exhaust Particles Trigger Calcium Response in Human Endothelial Cells via Aryl Hydrocarbon Receptor Non-Genomic Signalling

    doi: 10.3390/ijms19051429

    Figure Lengend Snippet: Origin and nature of n -Hex- and DCM-EOM-induced [Ca 2+ ] i responses. Cells were incubated in a buffer with or without Ca 2+ and ethylene glycol-bis(β-aminoethyl ether)- N , N , N ′, N ′-tetraacetic acid (EGTA), 30 min before exposure. Three min after measurements were started, the cells were exposed to n -Hex- ( A ) or DCM-EOM ( B ) at concentrations corresponding to 5 μg/mL of the original DEPs or vehicle control (DMSO). [Ca 2+ ] i level measured by normalized ratio of the Fura2-AM probe during exposure is presented as a graph and AUC from 1.0 at the Y -axis and 0–45 min, as mean and mean ± SEM ( n = 3), respectively. * Significantly different from calcium free exposure with EGTA ( p

    Article Snippet: 2-Aminoethoxydiphenylborate (2-APB), 2-methyl-2H-pyrazole-3-carboxylic acid (2-methyl-4-o-tolylazo-phenyl)-amide , cholesterol, and ethylene glycol-bis(β-aminoethyl ether)- N , N , N ′, N ′-tetraacetic acid (EGTA) were purchased from Sigma-Aldrich (now Merck; Darmstadt, Germany).

    Techniques: Incubation

    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