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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: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Pemphigus foliaceus IgG causes dissociation of desmoglein 1-containing junctions without blocking desmoglein 1 transinteraction"

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

Journal: Journal of Clinical Investigation

doi: 10.1172/JCI23475

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).
Figure Legend 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).

Techniques Used: Binding Assay, Incubation, Labeling

2) Product Images from "Novel genetically encoded fluorescent probes enable real-time detection of potassium in vitro and in vivo"

Article Title: Novel genetically encoded fluorescent probes enable real-time detection of potassium in vitro and in vivo

Journal: Nature Communications

doi: 10.1038/s41467-017-01615-z

Real-time imaging of intracellular K + fluxes. a Representative single cell K + (blue solid line) and Ca 2+ (red dashed line) response of an INS-1 cell expressing lc-LysM GEPII 1.0 and CAR-GECO1 upon cell depolarization using 70 mM KCl ( n = 9 independent measurements/32 cells/32 cells responded as demonstrated). b Columns represent maximal ∆FRET ratio signals ± SD of INS-1 cells expressing cytosolic lc-LysM GEPII 1.0 upon depolarization under control conditions (ctrl, white bar, n = 17/96), in the absence of extracellular Ca 2+ (1 mM EGTA, black bar, n = 7/50), in the presence of 15 mM TEA (blue bar, n = 6/50), in the presence of 100 µM nifedipine (green bar, n = 6/41), and in cells loaded with BAPTA-AM (red bar, n = 6/53). *** P
Figure Legend Snippet: Real-time imaging of intracellular K + fluxes. a Representative single cell K + (blue solid line) and Ca 2+ (red dashed line) response of an INS-1 cell expressing lc-LysM GEPII 1.0 and CAR-GECO1 upon cell depolarization using 70 mM KCl ( n = 9 independent measurements/32 cells/32 cells responded as demonstrated). b Columns represent maximal ∆FRET ratio signals ± SD of INS-1 cells expressing cytosolic lc-LysM GEPII 1.0 upon depolarization under control conditions (ctrl, white bar, n = 17/96), in the absence of extracellular Ca 2+ (1 mM EGTA, black bar, n = 7/50), in the presence of 15 mM TEA (blue bar, n = 6/50), in the presence of 100 µM nifedipine (green bar, n = 6/41), and in cells loaded with BAPTA-AM (red bar, n = 6/53). *** P

Techniques Used: Imaging, Expressing

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Patch Clamp:

Article Title: Differential pH Dynamics in Synaptic Vesicles From Intact Glutamatergic and GABAergic Synapses
Article Snippet: Electrophysiological experiments at elevated temperature with perfusion of the extracellular solution as described for live imaging experiments. .. Whole-cell patch clamp recordings were made with borosilicate glass pipettes (3–4 MΩ) filled with an internal solution containing (in mM): 136 KCl, 17.8 HEPES, 1 EGTA (Carl Roth), 4.6 MgCl2 , 4 Na2 ATP, 0.3 Na2 GTP (Sigma-Aldrich), 12 creatine phosphate (Calbiochem) and 50 U/ml phosphocreatine kinase (Sigma-Aldrich); 300 mOsm; pH 7.4. .. All recordings were performed with a Multiclamp 700B amplifier and Digidata 1440A under the control of Clampex 10 (all Molecular Devices).

Article Title: Self-organization of modular network architecture by activity-dependent neuronal migration and outgrowth
Article Snippet: .. Patch-clamp recording and analysis Patch pipettes (6.3 ± 1.4 MΩ) were filled with a intracellular solution, containing potassium D-gluconate (125 mM; Sigma-Aldrich), KCl (20 mM; Sigma-Aldrich), EGTA (5 mM; Carl Roth), Na2 -ATP (2 mM; Carl Roth), HEPES (10 mM; Carl Roth), MgCl2 (2 mM; Sigma-Aldrich) and CaCl2 (0.5 mM; Sigma-Aldrich), adjusted with KOH to pH 7.4, and with sucrose to 320 mOsm. .. Patch-clamp recordings in whole-cell configuration were conducted at 37°C (PH01 perfusion heating, MCS; TC02 temperature controller, MCS) and perfusion with carbogenated (95% O2 and 5% CO2 ; Air Liquide, Düsseldorf, Germany) culture medium without horse serum and without Gö6976 and PMA.

Transferring:

Article Title: Pathophysiological Consequences of Neuronal α-Synuclein Overexpression: Impacts on Ion Homeostasis, Stress Signaling, Mitochondrial Integrity, and Electrical Activity
Article Snippet: Silver wire electrodes were chlorinated using an ACL-01 apparatus (npi electronic) and a 2 M KCl solution. .. Patch pipettes were back-filled with 7 μl internal solution containing (in mM): 130 potassium gluconate, 8 KCl, 2 CaCl2 , 1 MgCl2 , 10 EGTA, 10 HEPES, 2 Mg-ATP, 0.3 GTP-Na (all from Roth; mOsm = 290-295, pH = 7.3), using 20 μl microloader pipette tips (Eppendorf; cat. no. 5242 956.003). .. Pipette holders were mounted on and controlled using micromanipulators from Scientifica.

Article Title: Investigation of Synapse Formation and Function in a Glutamatergic-GABAergic Two-Neuron Microcircuit
Article Snippet: Hypertonic solution for measuring readily releasable vesicle pool (RRP) size was made by adding 500 m m sucrose (Sigma-Aldrich) to the standard extracellular solution. .. The KCl patch pipette solution contained the following (in m m ): 136 KCl, 17.8 HEPES, and 1 EGTA (Carl Roth); 0.6 MgCl2 and 4 ATP-Mg (Sigma-Aldrich); 0.3 GTP-Na (Sigma-Aldrich); 12 phosphocreatine (Calbiochem, MERCK); and 50 U*ml−1 phosphocreatine kinase (Sigma-Aldrich), 300 mOsm, pH 7.4. ..

Article Title: β-Secretase BACE1 Promotes Surface Expression and Function of Kv3.4 at Hippocampal Mossy Fiber Synapses
Article Snippet: .. Patch electrodes were filled with a pipette solution containing the following (in m m ) (Sigma-Aldrich): 135 potassium gluconate, 5 HEPES, 3 MgCl2 , 5 EGTA, 2 Na2 -ATP, 0.3 Na3 -GTP, and 4 NaCl adjusted to pH 7.25 with KOH (Carl Roth). .. Cells were kept in bath solution containing the following (in m m ) (Sigma-Aldrich): 145 NaCl, 4 KCl, 2 MgCl2 , 2 CaCl2 , 10 HEPES, and 10 D(+)-glucose, adjusted to pH 7.4 with NaOH (Merck).

Dot Blot:

Article Title: Pemphigus foliaceus IgG causes dissociation of desmoglein 1-containing junctions without blocking desmoglein 1 transinteraction
Article Snippet: The cultures were used for all experiments when grown to confluent monolayers, and Dsg1 expression was detected by dot blot analysis (Figure ) as well as immunostaining (day 7 after plating) (see Figure A). .. For dot blot analysis, cells from a T25 tissue flask were harvested in 150 μl of HBSS following incubation for 20 minutes in PBS consisting of 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2 HPO4 , and 1.5 mM KH2PO4, pH 7.4) containing 1% EGTA (Roth), followed by 10 minutes of incubation in EGTA/trypsin (0.025%/0.05% final concentration each). ..

Incubation:

Article Title: Pemphigus foliaceus IgG causes dissociation of desmoglein 1-containing junctions without blocking desmoglein 1 transinteraction
Article Snippet: The cultures were used for all experiments when grown to confluent monolayers, and Dsg1 expression was detected by dot blot analysis (Figure ) as well as immunostaining (day 7 after plating) (see Figure A). .. For dot blot analysis, cells from a T25 tissue flask were harvested in 150 μl of HBSS following incubation for 20 minutes in PBS consisting of 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2 HPO4 , and 1.5 mM KH2PO4, pH 7.4) containing 1% EGTA (Roth), followed by 10 minutes of incubation in EGTA/trypsin (0.025%/0.05% final concentration each). ..

Article Title: Neuronal Dysfunction in iPSC-Derived Medium Spiny Neurons from Chorea-Acanthocytosis Patients Is Reversed by Src Kinase Inhibition and F-Actin Stabilization
Article Snippet: Mature neurons were washed with ice-cold PBS containing phosphatase inhibitor (Thermo Fisher Scientific). .. Cells were then incubated with extraction buffer consisting of 0.3% Triton X-100 (Thermo Fisher Scientific), 5 m m Tris/HCl (Roth), pH 7.4, 2 m m EGTA (Roth), 300 m m sucrose (Roth), 2 μ m phalloidin (Tocris Bioscience), and 1:100 protease inhibitor (Thermo Fisher Scientific) for 5 min on ice. .. The supernatant was collected and the same volume of RIPA lysis buffer was applied containing 50 m m Tris/HCl, pH 7.4, 1% Triton X-100, 1% sodium deoxycholate (Sigma-Aldrich), 0.1% SDS (Roth), 0.15 m NaCl (Roth), 1 m m EDTA (Roth), 1 m m DTT (Roth), and 1 m m sodium orthovanadate (Sigma-Aldrich).

Concentration Assay:

Article Title: Pemphigus foliaceus IgG causes dissociation of desmoglein 1-containing junctions without blocking desmoglein 1 transinteraction
Article Snippet: The cultures were used for all experiments when grown to confluent monolayers, and Dsg1 expression was detected by dot blot analysis (Figure ) as well as immunostaining (day 7 after plating) (see Figure A). .. For dot blot analysis, cells from a T25 tissue flask were harvested in 150 μl of HBSS following incubation for 20 minutes in PBS consisting of 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2 HPO4 , and 1.5 mM KH2PO4, pH 7.4) containing 1% EGTA (Roth), followed by 10 minutes of incubation in EGTA/trypsin (0.025%/0.05% final concentration each). ..

Protease Inhibitor:

Article Title: Neuronal Dysfunction in iPSC-Derived Medium Spiny Neurons from Chorea-Acanthocytosis Patients Is Reversed by Src Kinase Inhibition and F-Actin Stabilization
Article Snippet: Mature neurons were washed with ice-cold PBS containing phosphatase inhibitor (Thermo Fisher Scientific). .. Cells were then incubated with extraction buffer consisting of 0.3% Triton X-100 (Thermo Fisher Scientific), 5 m m Tris/HCl (Roth), pH 7.4, 2 m m EGTA (Roth), 300 m m sucrose (Roth), 2 μ m phalloidin (Tocris Bioscience), and 1:100 protease inhibitor (Thermo Fisher Scientific) for 5 min on ice. .. The supernatant was collected and the same volume of RIPA lysis buffer was applied containing 50 m m Tris/HCl, pH 7.4, 1% Triton X-100, 1% sodium deoxycholate (Sigma-Aldrich), 0.1% SDS (Roth), 0.15 m NaCl (Roth), 1 m m EDTA (Roth), 1 m m DTT (Roth), and 1 m m sodium orthovanadate (Sigma-Aldrich).

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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).
    Egta, supplied by Carl Roth GmbH, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/egta/product/Carl Roth GmbH
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    egta - by Bioz Stars, 2021-03
    86/100 stars
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    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