pctx1  (Alomone Labs)


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    Alomone Labs pctx1
    Racemic lactate triggers ASIC1a dependent [Ca 2+ ] c and [Ca 2+ ] m signals. (A) Illustration of the figure hypothesis; (B) Representative Fura-2 ratio traces for [Ca 2+ ] c changes monitored in WT primary cultured hippocampal neurons (DIV 10–15) without and with pretreatment of the selective ASIC1a inhibitor <t>PcTx1</t> (20 nM, 120 s). Neurons were loaded with Fura-2AM (1 μM) and initially superfused with Ringer's solution at pH 7.4. Then, the superfusion was switched to Ringer's solution of pH 7.4 with an addition of DL-lactate (5 mM) as indicated by the arrowhead; (C) Quantification of the number of cytosolic (cyto) Ca 2+ peaks (transients) per 180-s time period measured as in (B) for WT neurons untreated (n = 39) and treated (n = 77); Box and whiskers plot show maximal and minimal values and all data points; (D) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in Rhod-2 AM (1 μM)-loaded WT neurons untreated and treated with PcTX1. Superfusion was switched to pH 7.4 Ringer's solution with DL-lactate; (E) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (D) for WT neurons untreated (n = 11) and treated with PcTX1 (n = 65); (F) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in response to direct puffing of the pH 7.4-DL- lactate solution in primary cultured WT and KO cortical neurons; (G) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (F) for WT (n = 6) and KO cortical neurons (n = 5); All summary data represent mean ± SD, ****p < 0.0001.
    Pctx1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pctx1/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pctx1 - by Bioz Stars, 2023-03
    93/100 stars

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    1) Product Images from "ASIC1a senses lactate uptake to regulate metabolism in neurons"

    Article Title: ASIC1a senses lactate uptake to regulate metabolism in neurons

    Journal: Redox Biology

    doi: 10.1016/j.redox.2022.102253

    Racemic lactate triggers ASIC1a dependent [Ca 2+ ] c and [Ca 2+ ] m signals. (A) Illustration of the figure hypothesis; (B) Representative Fura-2 ratio traces for [Ca 2+ ] c changes monitored in WT primary cultured hippocampal neurons (DIV 10–15) without and with pretreatment of the selective ASIC1a inhibitor PcTx1 (20 nM, 120 s). Neurons were loaded with Fura-2AM (1 μM) and initially superfused with Ringer's solution at pH 7.4. Then, the superfusion was switched to Ringer's solution of pH 7.4 with an addition of DL-lactate (5 mM) as indicated by the arrowhead; (C) Quantification of the number of cytosolic (cyto) Ca 2+ peaks (transients) per 180-s time period measured as in (B) for WT neurons untreated (n = 39) and treated (n = 77); Box and whiskers plot show maximal and minimal values and all data points; (D) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in Rhod-2 AM (1 μM)-loaded WT neurons untreated and treated with PcTX1. Superfusion was switched to pH 7.4 Ringer's solution with DL-lactate; (E) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (D) for WT neurons untreated (n = 11) and treated with PcTX1 (n = 65); (F) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in response to direct puffing of the pH 7.4-DL- lactate solution in primary cultured WT and KO cortical neurons; (G) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (F) for WT (n = 6) and KO cortical neurons (n = 5); All summary data represent mean ± SD, ****p < 0.0001.
    Figure Legend Snippet: Racemic lactate triggers ASIC1a dependent [Ca 2+ ] c and [Ca 2+ ] m signals. (A) Illustration of the figure hypothesis; (B) Representative Fura-2 ratio traces for [Ca 2+ ] c changes monitored in WT primary cultured hippocampal neurons (DIV 10–15) without and with pretreatment of the selective ASIC1a inhibitor PcTx1 (20 nM, 120 s). Neurons were loaded with Fura-2AM (1 μM) and initially superfused with Ringer's solution at pH 7.4. Then, the superfusion was switched to Ringer's solution of pH 7.4 with an addition of DL-lactate (5 mM) as indicated by the arrowhead; (C) Quantification of the number of cytosolic (cyto) Ca 2+ peaks (transients) per 180-s time period measured as in (B) for WT neurons untreated (n = 39) and treated (n = 77); Box and whiskers plot show maximal and minimal values and all data points; (D) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in Rhod-2 AM (1 μM)-loaded WT neurons untreated and treated with PcTX1. Superfusion was switched to pH 7.4 Ringer's solution with DL-lactate; (E) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (D) for WT neurons untreated (n = 11) and treated with PcTX1 (n = 65); (F) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in response to direct puffing of the pH 7.4-DL- lactate solution in primary cultured WT and KO cortical neurons; (G) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (F) for WT (n = 6) and KO cortical neurons (n = 5); All summary data represent mean ± SD, ****p < 0.0001.

    Techniques Used: Cell Culture, Fluorescence

    ASIC1a mediates L -lactate-induced increase in mitochondrial respiration and suppresses mitochondrial ROS production . Seahorse analysis (see Materials and Methods) was used to monitor mitochondrial respiration (OCR) with sequential additions of oligomycin (1 μM), FCCP (1 μM) + sodium pyruvate (5 mM), and a mix of rotenone/antimycin A (Ro/AA, 0.5 μM each), as indicated by the arrowheads, in media that contained or not D-, or L-lactate (5 mM); (A) Representative OCR plots of WT neurons in regular medium that contained no lactate (Ctrl) or the indicated lactate isomer and CIN4; (B) Quantification of maximal respiration as measured in (A) of WT neurons in regular medium (n = 6), and the medium that contained D- (n = 6) or L-lactate (n = 6), or L-lactate plus CIN4 (n = 6); (C) Representative OCR plots of KO neurons in regular medium or medium that contained the indicated lactate isomer and CIN4; (D) Quantification of maximal respiration as measured in (C) of KO in regular medium (Ctrl, n = 6) and media that contained the indicated D-lactate (n = 6), L-lactate (n-6), and L-lactate + CIN4 (n = 6); (E) Representative OCR plots of WT and KO neurons in L -lactate containing medium; (F) Quantification of maximal respiration as measured in (E) of WT (n = 6) and KO (n = 6) neurons; (G) Representative OCR plots of WT and KO neurons in L-lactate/CIN4 containing medium (n = 6); (H) Quantification of maximal respiration as measured in (G) of WT and KO neurons in L -lactate/CIN4 containing medium (n = 6 for each); (I) Representative RoGFP fluorescence traces for redox changes in response to L-lactate, H 2 O 2 and DTT added in the Ringer's solution in WT neurons untreated and treated with PcTX1; (J) Quantification of R/R 0 (480/405) at 500s after the addition of L-lactate (100s) as in (I) for WT neurons untreated (n = 9) and treated with PcTX1 (n = 7); (K) Schematic presentation of suggested pathway linking L-lactate to ASIC1a. All summary graph data represent mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.
    Figure Legend Snippet: ASIC1a mediates L -lactate-induced increase in mitochondrial respiration and suppresses mitochondrial ROS production . Seahorse analysis (see Materials and Methods) was used to monitor mitochondrial respiration (OCR) with sequential additions of oligomycin (1 μM), FCCP (1 μM) + sodium pyruvate (5 mM), and a mix of rotenone/antimycin A (Ro/AA, 0.5 μM each), as indicated by the arrowheads, in media that contained or not D-, or L-lactate (5 mM); (A) Representative OCR plots of WT neurons in regular medium that contained no lactate (Ctrl) or the indicated lactate isomer and CIN4; (B) Quantification of maximal respiration as measured in (A) of WT neurons in regular medium (n = 6), and the medium that contained D- (n = 6) or L-lactate (n = 6), or L-lactate plus CIN4 (n = 6); (C) Representative OCR plots of KO neurons in regular medium or medium that contained the indicated lactate isomer and CIN4; (D) Quantification of maximal respiration as measured in (C) of KO in regular medium (Ctrl, n = 6) and media that contained the indicated D-lactate (n = 6), L-lactate (n-6), and L-lactate + CIN4 (n = 6); (E) Representative OCR plots of WT and KO neurons in L -lactate containing medium; (F) Quantification of maximal respiration as measured in (E) of WT (n = 6) and KO (n = 6) neurons; (G) Representative OCR plots of WT and KO neurons in L-lactate/CIN4 containing medium (n = 6); (H) Quantification of maximal respiration as measured in (G) of WT and KO neurons in L -lactate/CIN4 containing medium (n = 6 for each); (I) Representative RoGFP fluorescence traces for redox changes in response to L-lactate, H 2 O 2 and DTT added in the Ringer's solution in WT neurons untreated and treated with PcTX1; (J) Quantification of R/R 0 (480/405) at 500s after the addition of L-lactate (100s) as in (I) for WT neurons untreated (n = 9) and treated with PcTX1 (n = 7); (K) Schematic presentation of suggested pathway linking L-lactate to ASIC1a. All summary graph data represent mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

    Techniques Used: Fluorescence

    pctx1  (Alomone Labs)


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    Alomone Labs pctx1
    Racemic lactate triggers ASIC1a dependent [Ca 2+ ] c and [Ca 2+ ] m signals. (A) Illustration of the figure hypothesis; (B) Representative Fura-2 ratio traces for [Ca 2+ ] c changes monitored in WT primary cultured hippocampal neurons (DIV 10–15) without and with pretreatment of the selective ASIC1a inhibitor <t>PcTx1</t> (20 nM, 120 s). Neurons were loaded with Fura-2AM (1 μM) and initially superfused with Ringer's solution at pH 7.4. Then, the superfusion was switched to Ringer's solution of pH 7.4 with an addition of DL-lactate (5 mM) as indicated by the arrowhead; (C) Quantification of the number of cytosolic (cyto) Ca 2+ peaks (transients) per 180-s time period measured as in (B) for WT neurons untreated (n = 39) and treated (n = 77); Box and whiskers plot show maximal and minimal values and all data points; (D) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in Rhod-2 AM (1 μM)-loaded WT neurons untreated and treated with PcTX1. Superfusion was switched to pH 7.4 Ringer's solution with DL-lactate; (E) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (D) for WT neurons untreated (n = 11) and treated with PcTX1 (n = 65); (F) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in response to direct puffing of the pH 7.4-DL- lactate solution in primary cultured WT and KO cortical neurons; (G) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (F) for WT (n = 6) and KO cortical neurons (n = 5); All summary data represent mean ± SD, ****p < 0.0001.
    Pctx1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pctx1/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pctx1 - by Bioz Stars, 2023-03
    93/100 stars

    Images

    1) Product Images from "ASIC1a senses lactate uptake to regulate metabolism in neurons"

    Article Title: ASIC1a senses lactate uptake to regulate metabolism in neurons

    Journal: Redox Biology

    doi: 10.1016/j.redox.2022.102253

    Racemic lactate triggers ASIC1a dependent [Ca 2+ ] c and [Ca 2+ ] m signals. (A) Illustration of the figure hypothesis; (B) Representative Fura-2 ratio traces for [Ca 2+ ] c changes monitored in WT primary cultured hippocampal neurons (DIV 10–15) without and with pretreatment of the selective ASIC1a inhibitor PcTx1 (20 nM, 120 s). Neurons were loaded with Fura-2AM (1 μM) and initially superfused with Ringer's solution at pH 7.4. Then, the superfusion was switched to Ringer's solution of pH 7.4 with an addition of DL-lactate (5 mM) as indicated by the arrowhead; (C) Quantification of the number of cytosolic (cyto) Ca 2+ peaks (transients) per 180-s time period measured as in (B) for WT neurons untreated (n = 39) and treated (n = 77); Box and whiskers plot show maximal and minimal values and all data points; (D) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in Rhod-2 AM (1 μM)-loaded WT neurons untreated and treated with PcTX1. Superfusion was switched to pH 7.4 Ringer's solution with DL-lactate; (E) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (D) for WT neurons untreated (n = 11) and treated with PcTX1 (n = 65); (F) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in response to direct puffing of the pH 7.4-DL- lactate solution in primary cultured WT and KO cortical neurons; (G) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (F) for WT (n = 6) and KO cortical neurons (n = 5); All summary data represent mean ± SD, ****p < 0.0001.
    Figure Legend Snippet: Racemic lactate triggers ASIC1a dependent [Ca 2+ ] c and [Ca 2+ ] m signals. (A) Illustration of the figure hypothesis; (B) Representative Fura-2 ratio traces for [Ca 2+ ] c changes monitored in WT primary cultured hippocampal neurons (DIV 10–15) without and with pretreatment of the selective ASIC1a inhibitor PcTx1 (20 nM, 120 s). Neurons were loaded with Fura-2AM (1 μM) and initially superfused with Ringer's solution at pH 7.4. Then, the superfusion was switched to Ringer's solution of pH 7.4 with an addition of DL-lactate (5 mM) as indicated by the arrowhead; (C) Quantification of the number of cytosolic (cyto) Ca 2+ peaks (transients) per 180-s time period measured as in (B) for WT neurons untreated (n = 39) and treated (n = 77); Box and whiskers plot show maximal and minimal values and all data points; (D) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in Rhod-2 AM (1 μM)-loaded WT neurons untreated and treated with PcTX1. Superfusion was switched to pH 7.4 Ringer's solution with DL-lactate; (E) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (D) for WT neurons untreated (n = 11) and treated with PcTX1 (n = 65); (F) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in response to direct puffing of the pH 7.4-DL- lactate solution in primary cultured WT and KO cortical neurons; (G) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (F) for WT (n = 6) and KO cortical neurons (n = 5); All summary data represent mean ± SD, ****p < 0.0001.

    Techniques Used: Cell Culture, Fluorescence

    ASIC1a mediates L -lactate-induced increase in mitochondrial respiration and suppresses mitochondrial ROS production . Seahorse analysis (see Materials and Methods) was used to monitor mitochondrial respiration (OCR) with sequential additions of oligomycin (1 μM), FCCP (1 μM) + sodium pyruvate (5 mM), and a mix of rotenone/antimycin A (Ro/AA, 0.5 μM each), as indicated by the arrowheads, in media that contained or not D-, or L-lactate (5 mM); (A) Representative OCR plots of WT neurons in regular medium that contained no lactate (Ctrl) or the indicated lactate isomer and CIN4; (B) Quantification of maximal respiration as measured in (A) of WT neurons in regular medium (n = 6), and the medium that contained D- (n = 6) or L-lactate (n = 6), or L-lactate plus CIN4 (n = 6); (C) Representative OCR plots of KO neurons in regular medium or medium that contained the indicated lactate isomer and CIN4; (D) Quantification of maximal respiration as measured in (C) of KO in regular medium (Ctrl, n = 6) and media that contained the indicated D-lactate (n = 6), L-lactate (n-6), and L-lactate + CIN4 (n = 6); (E) Representative OCR plots of WT and KO neurons in L -lactate containing medium; (F) Quantification of maximal respiration as measured in (E) of WT (n = 6) and KO (n = 6) neurons; (G) Representative OCR plots of WT and KO neurons in L-lactate/CIN4 containing medium (n = 6); (H) Quantification of maximal respiration as measured in (G) of WT and KO neurons in L -lactate/CIN4 containing medium (n = 6 for each); (I) Representative RoGFP fluorescence traces for redox changes in response to L-lactate, H 2 O 2 and DTT added in the Ringer's solution in WT neurons untreated and treated with PcTX1; (J) Quantification of R/R 0 (480/405) at 500s after the addition of L-lactate (100s) as in (I) for WT neurons untreated (n = 9) and treated with PcTX1 (n = 7); (K) Schematic presentation of suggested pathway linking L-lactate to ASIC1a. All summary graph data represent mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.
    Figure Legend Snippet: ASIC1a mediates L -lactate-induced increase in mitochondrial respiration and suppresses mitochondrial ROS production . Seahorse analysis (see Materials and Methods) was used to monitor mitochondrial respiration (OCR) with sequential additions of oligomycin (1 μM), FCCP (1 μM) + sodium pyruvate (5 mM), and a mix of rotenone/antimycin A (Ro/AA, 0.5 μM each), as indicated by the arrowheads, in media that contained or not D-, or L-lactate (5 mM); (A) Representative OCR plots of WT neurons in regular medium that contained no lactate (Ctrl) or the indicated lactate isomer and CIN4; (B) Quantification of maximal respiration as measured in (A) of WT neurons in regular medium (n = 6), and the medium that contained D- (n = 6) or L-lactate (n = 6), or L-lactate plus CIN4 (n = 6); (C) Representative OCR plots of KO neurons in regular medium or medium that contained the indicated lactate isomer and CIN4; (D) Quantification of maximal respiration as measured in (C) of KO in regular medium (Ctrl, n = 6) and media that contained the indicated D-lactate (n = 6), L-lactate (n-6), and L-lactate + CIN4 (n = 6); (E) Representative OCR plots of WT and KO neurons in L -lactate containing medium; (F) Quantification of maximal respiration as measured in (E) of WT (n = 6) and KO (n = 6) neurons; (G) Representative OCR plots of WT and KO neurons in L-lactate/CIN4 containing medium (n = 6); (H) Quantification of maximal respiration as measured in (G) of WT and KO neurons in L -lactate/CIN4 containing medium (n = 6 for each); (I) Representative RoGFP fluorescence traces for redox changes in response to L-lactate, H 2 O 2 and DTT added in the Ringer's solution in WT neurons untreated and treated with PcTX1; (J) Quantification of R/R 0 (480/405) at 500s after the addition of L-lactate (100s) as in (I) for WT neurons untreated (n = 9) and treated with PcTX1 (n = 7); (K) Schematic presentation of suggested pathway linking L-lactate to ASIC1a. All summary graph data represent mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

    Techniques Used: Fluorescence

    psalmotoxin 1 pctx1  (Alomone Labs)


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    Alomone Labs psalmotoxin 1 pctx1
    Psalmotoxin 1 Pctx1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/psalmotoxin 1 pctx1/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
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    pctx1  (Alomone Labs)


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    Alomone Labs pctx1
    (A) Representative fluorescence traces of cytosolic Na+ concentration ([Na+]c) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor <t>PcTX1</t> (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated.
    Pctx1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pctx1/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pctx1 - by Bioz Stars, 2023-03
    93/100 stars

    Images

    1) Product Images from "ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons"

    Article Title: ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons

    Journal: Journal of neurochemistry

    doi: 10.1111/jnc.14971

    (A) Representative fluorescence traces of cytosolic Na+ concentration ([Na+]c) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated.
    Figure Legend Snippet: (A) Representative fluorescence traces of cytosolic Na+ concentration ([Na+]c) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated.

    Techniques Used: Fluorescence, Concentration Assay, Cell Culture

    (A) Representative [Na+]m fluorescence traces in digitonin-permeabilized HEK293-T cells overexpressing ASIC1a (See Materials and Methods). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion.
    Figure Legend Snippet: (A) Representative [Na+]m fluorescence traces in digitonin-permeabilized HEK293-T cells overexpressing ASIC1a (See Materials and Methods). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion.

    Techniques Used: Fluorescence

    A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca2+ concentration ([Ca2+]c) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion.
    Figure Legend Snippet: A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca2+ concentration ([Ca2+]c) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion.

    Techniques Used: Fluorescence, Concentration Assay

    pctx1  (Alomone Labs)


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    Alomone Labs pctx1
    (A) Representative fluorescence traces of cytosolic Na+ concentration ([Na+]c) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor <t>PcTX1</t> (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated.
    Pctx1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pctx1/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pctx1 - by Bioz Stars, 2023-03
    93/100 stars

    Images

    1) Product Images from "ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons"

    Article Title: ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons

    Journal: Journal of neurochemistry

    doi: 10.1111/jnc.14971

    (A) Representative fluorescence traces of cytosolic Na+ concentration ([Na+]c) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated.
    Figure Legend Snippet: (A) Representative fluorescence traces of cytosolic Na+ concentration ([Na+]c) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated.

    Techniques Used: Fluorescence, Concentration Assay, Cell Culture

    (A) Representative [Na+]m fluorescence traces in digitonin-permeabilized HEK293-T cells overexpressing ASIC1a (See Materials and Methods). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion.
    Figure Legend Snippet: (A) Representative [Na+]m fluorescence traces in digitonin-permeabilized HEK293-T cells overexpressing ASIC1a (See Materials and Methods). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion.

    Techniques Used: Fluorescence

    A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca2+ concentration ([Ca2+]c) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion.
    Figure Legend Snippet: A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca2+ concentration ([Ca2+]c) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion.

    Techniques Used: Fluorescence, Concentration Assay

    psalmotoxin 1 pctx1  (Alomone Labs)


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    Structured Review

    Alomone Labs psalmotoxin 1 pctx1
    A role for ASIC1b in acid-induced mechanical hyperalgesia. (A) Mechanical hyperalgesia of wild-type ( Asic1b + / + , N = 7) and ASIC1b-knockout ( Asic1b – /– , N = 8) mice was induced by intramuscular injections of pH-4.0 saline on days 0 and 1 and evaluated by 0.20-mN von Frey filament. Data were analyzed by two-way ANOVA (Interaction F ( 10 , 143 ) = 5.34, P < 0.0001; Time F ( 10 , 143 ) = 14.5, P < 0.0001; Genotype F ( 1 , 143 ) = 23.11, P < 0.0001), followed by Sidak post hoc test. ∗∗∗ P < 0.001 for genotype difference at specific times. (B) Mechanical hyperalgesia of Asic1b + / + ( N = 10) and Asic1b – /– ( N = 7) mice was induced by intramuscular injections of pH-4.0 saline on days 0 and 5. Data were analyzed by two-way ANOVA [Interaction F ( 12 , 195 ) = 5.933, P < 0.0001; Time F ( 12 , 195 ) = 21.28, P < 0.0001; Genotype F ( 1 , 195 ) = 59.07, P < 0.0001], followed by Sidak post hoc test. **** P < 0.0001; ∗∗∗ P < 0.001; ∗∗ P < 0.01; ∗ P < 0.05 Asic1b + / + vs. Asic1b – /– . For comparison, the effect of mambalgin-1 on acid-induced hyperalgesia in Asic1b – /– mice was plotted. (C) Effect of mambalgin-1 (MB-1) on acid-induced mechanical hyperalgesia (vehicle, N = 7; MB-1 [3 pmol], N = 6; MB-1 [15 pmol], N = 7; MB-1 [30 pmol], N = 7). Data were analyzed by two-way ANOVA [Interaction F ( 21 , 168 ) = 4.014, P < 0.0001; Time F ( 7 , 168 ) = 26.61, P < 0.0001; Drug dose F ( 3 , 168 ) = 29.32, P < 0.0001], followed by Sidak post hoc test. ### P < 0.001, #### P < 0.0001, MB-1 (30 pmol) vs. vehicle; $$$$ P < 0.0001, MB-1 (15 pmol) vs. vehicle. (D) Cumulative withdrawal response after second acid injection 5 days before acid + MB-1 injection is shown as the area under the receiver operating characteristic curve (AUC) calculated by the trapezoidal method. Data were analyzed by one-way ANOVA [ F ( 3 , 21 ) = 4.719, P = 0.0114], followed by Dunnett post hoc test. ∗∗ P < 0.01, ∗ P < 0.05 vs. vehicle. (D’) While 30 pmole mambalgin-1 was applied in the second acid injection, it blocked the development of the acid-induced chronic hyperalgesia. (E) Effect of <t>PcTx1</t> on acid-induced mechanical hyperalgesia. Data were analyzed by two-way ANOVA (vehicle, N = 4; PcTx1 [120 nmol], N = 6) Interaction F ( 7 , 64 ) = 0.2935, P = 0.9541; Time F ( 7 , 64 ) = 31.29, P < 0.0001; Drug dose F ( 1 , 64 ) = 0.054, P = 0.8169). (F) Cumulative withdrawal response after a second acid injection 5 days before acid + PcTx1 injection is shown as the AUC calculated by the trapezoidal method. Data were analyzed by unpaired t -test [ F ( 3 , 5 ) = 2.191, P = 0.4149). Black arrows, the time mice received intramuscular injection of pH 4.0 saline. Red arrows, the time mice received intramuscular injection of pH-4.0 saline mixed with mambalgin-1 or PcTx1. B, baseline. Data are mean ± SEM.
    Psalmotoxin 1 Pctx1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Involvement of Acid-Sensing Ion Channel 1b in the Development of Acid-Induced Chronic Muscle Pain"

    Article Title: Involvement of Acid-Sensing Ion Channel 1b in the Development of Acid-Induced Chronic Muscle Pain

    Journal: Frontiers in Neuroscience

    doi: 10.3389/fnins.2019.01247

    A role for ASIC1b in acid-induced mechanical hyperalgesia. (A) Mechanical hyperalgesia of wild-type ( Asic1b + / + , N = 7) and ASIC1b-knockout ( Asic1b – /– , N = 8) mice was induced by intramuscular injections of pH-4.0 saline on days 0 and 1 and evaluated by 0.20-mN von Frey filament. Data were analyzed by two-way ANOVA (Interaction F ( 10 , 143 ) = 5.34, P < 0.0001; Time F ( 10 , 143 ) = 14.5, P < 0.0001; Genotype F ( 1 , 143 ) = 23.11, P < 0.0001), followed by Sidak post hoc test. ∗∗∗ P < 0.001 for genotype difference at specific times. (B) Mechanical hyperalgesia of Asic1b + / + ( N = 10) and Asic1b – /– ( N = 7) mice was induced by intramuscular injections of pH-4.0 saline on days 0 and 5. Data were analyzed by two-way ANOVA [Interaction F ( 12 , 195 ) = 5.933, P < 0.0001; Time F ( 12 , 195 ) = 21.28, P < 0.0001; Genotype F ( 1 , 195 ) = 59.07, P < 0.0001], followed by Sidak post hoc test. **** P < 0.0001; ∗∗∗ P < 0.001; ∗∗ P < 0.01; ∗ P < 0.05 Asic1b + / + vs. Asic1b – /– . For comparison, the effect of mambalgin-1 on acid-induced hyperalgesia in Asic1b – /– mice was plotted. (C) Effect of mambalgin-1 (MB-1) on acid-induced mechanical hyperalgesia (vehicle, N = 7; MB-1 [3 pmol], N = 6; MB-1 [15 pmol], N = 7; MB-1 [30 pmol], N = 7). Data were analyzed by two-way ANOVA [Interaction F ( 21 , 168 ) = 4.014, P < 0.0001; Time F ( 7 , 168 ) = 26.61, P < 0.0001; Drug dose F ( 3 , 168 ) = 29.32, P < 0.0001], followed by Sidak post hoc test. ### P < 0.001, #### P < 0.0001, MB-1 (30 pmol) vs. vehicle; $$$$ P < 0.0001, MB-1 (15 pmol) vs. vehicle. (D) Cumulative withdrawal response after second acid injection 5 days before acid + MB-1 injection is shown as the area under the receiver operating characteristic curve (AUC) calculated by the trapezoidal method. Data were analyzed by one-way ANOVA [ F ( 3 , 21 ) = 4.719, P = 0.0114], followed by Dunnett post hoc test. ∗∗ P < 0.01, ∗ P < 0.05 vs. vehicle. (D’) While 30 pmole mambalgin-1 was applied in the second acid injection, it blocked the development of the acid-induced chronic hyperalgesia. (E) Effect of PcTx1 on acid-induced mechanical hyperalgesia. Data were analyzed by two-way ANOVA (vehicle, N = 4; PcTx1 [120 nmol], N = 6) Interaction F ( 7 , 64 ) = 0.2935, P = 0.9541; Time F ( 7 , 64 ) = 31.29, P < 0.0001; Drug dose F ( 1 , 64 ) = 0.054, P = 0.8169). (F) Cumulative withdrawal response after a second acid injection 5 days before acid + PcTx1 injection is shown as the AUC calculated by the trapezoidal method. Data were analyzed by unpaired t -test [ F ( 3 , 5 ) = 2.191, P = 0.4149). Black arrows, the time mice received intramuscular injection of pH 4.0 saline. Red arrows, the time mice received intramuscular injection of pH-4.0 saline mixed with mambalgin-1 or PcTx1. B, baseline. Data are mean ± SEM.
    Figure Legend Snippet: A role for ASIC1b in acid-induced mechanical hyperalgesia. (A) Mechanical hyperalgesia of wild-type ( Asic1b + / + , N = 7) and ASIC1b-knockout ( Asic1b – /– , N = 8) mice was induced by intramuscular injections of pH-4.0 saline on days 0 and 1 and evaluated by 0.20-mN von Frey filament. Data were analyzed by two-way ANOVA (Interaction F ( 10 , 143 ) = 5.34, P < 0.0001; Time F ( 10 , 143 ) = 14.5, P < 0.0001; Genotype F ( 1 , 143 ) = 23.11, P < 0.0001), followed by Sidak post hoc test. ∗∗∗ P < 0.001 for genotype difference at specific times. (B) Mechanical hyperalgesia of Asic1b + / + ( N = 10) and Asic1b – /– ( N = 7) mice was induced by intramuscular injections of pH-4.0 saline on days 0 and 5. Data were analyzed by two-way ANOVA [Interaction F ( 12 , 195 ) = 5.933, P < 0.0001; Time F ( 12 , 195 ) = 21.28, P < 0.0001; Genotype F ( 1 , 195 ) = 59.07, P < 0.0001], followed by Sidak post hoc test. **** P < 0.0001; ∗∗∗ P < 0.001; ∗∗ P < 0.01; ∗ P < 0.05 Asic1b + / + vs. Asic1b – /– . For comparison, the effect of mambalgin-1 on acid-induced hyperalgesia in Asic1b – /– mice was plotted. (C) Effect of mambalgin-1 (MB-1) on acid-induced mechanical hyperalgesia (vehicle, N = 7; MB-1 [3 pmol], N = 6; MB-1 [15 pmol], N = 7; MB-1 [30 pmol], N = 7). Data were analyzed by two-way ANOVA [Interaction F ( 21 , 168 ) = 4.014, P < 0.0001; Time F ( 7 , 168 ) = 26.61, P < 0.0001; Drug dose F ( 3 , 168 ) = 29.32, P < 0.0001], followed by Sidak post hoc test. ### P < 0.001, #### P < 0.0001, MB-1 (30 pmol) vs. vehicle; $$$$ P < 0.0001, MB-1 (15 pmol) vs. vehicle. (D) Cumulative withdrawal response after second acid injection 5 days before acid + MB-1 injection is shown as the area under the receiver operating characteristic curve (AUC) calculated by the trapezoidal method. Data were analyzed by one-way ANOVA [ F ( 3 , 21 ) = 4.719, P = 0.0114], followed by Dunnett post hoc test. ∗∗ P < 0.01, ∗ P < 0.05 vs. vehicle. (D’) While 30 pmole mambalgin-1 was applied in the second acid injection, it blocked the development of the acid-induced chronic hyperalgesia. (E) Effect of PcTx1 on acid-induced mechanical hyperalgesia. Data were analyzed by two-way ANOVA (vehicle, N = 4; PcTx1 [120 nmol], N = 6) Interaction F ( 7 , 64 ) = 0.2935, P = 0.9541; Time F ( 7 , 64 ) = 31.29, P < 0.0001; Drug dose F ( 1 , 64 ) = 0.054, P = 0.8169). (F) Cumulative withdrawal response after a second acid injection 5 days before acid + PcTx1 injection is shown as the AUC calculated by the trapezoidal method. Data were analyzed by unpaired t -test [ F ( 3 , 5 ) = 2.191, P = 0.4149). Black arrows, the time mice received intramuscular injection of pH 4.0 saline. Red arrows, the time mice received intramuscular injection of pH-4.0 saline mixed with mambalgin-1 or PcTx1. B, baseline. Data are mean ± SEM.

    Techniques Used: Knock-Out, Injection

    Electrophysiological properties of three types of ASIC1b-TdTomato(+) DRGs with different desensitization rates.
    Figure Legend Snippet: Electrophysiological properties of three types of ASIC1b-TdTomato(+) DRGs with different desensitization rates.

    Techniques Used: Inhibition

    Effect of amiloride, mambalgin-1, APETx2 and PcTx1 on ASIC1b-expressing muscle afferent DRG neurons. (A) Whole-cell patch clamp recording on an ASIC1b-expressing DRG neuron projecting to gastrocnemius muscle labeled by fluorogold. (B) Mambalgin-1 (MB-1) (1 μM) inhibited acid (pH 5.0)-induced currents in 13 of 14 ASIC1b-expressing muscle afferent DRG neurons. (C) APETx2 (1 μM) inhibited acid (pH 5.0)-induced currents in 6 of 13 ASIC1b-expressing muscle afferent DRG neurons. (D) PcTx1 (100 nM) inhibited acid (pH 5.0)-induced currents in 5 of 11 ASIC1b-expressing muscle afferent DRG neurons.
    Figure Legend Snippet: Effect of amiloride, mambalgin-1, APETx2 and PcTx1 on ASIC1b-expressing muscle afferent DRG neurons. (A) Whole-cell patch clamp recording on an ASIC1b-expressing DRG neuron projecting to gastrocnemius muscle labeled by fluorogold. (B) Mambalgin-1 (MB-1) (1 μM) inhibited acid (pH 5.0)-induced currents in 13 of 14 ASIC1b-expressing muscle afferent DRG neurons. (C) APETx2 (1 μM) inhibited acid (pH 5.0)-induced currents in 6 of 13 ASIC1b-expressing muscle afferent DRG neurons. (D) PcTx1 (100 nM) inhibited acid (pH 5.0)-induced currents in 5 of 11 ASIC1b-expressing muscle afferent DRG neurons.

    Techniques Used: Expressing, Patch Clamp, Labeling

    psalmotoxin 1 pctx1  (Alomone Labs)


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    Alomone Labs psalmotoxin 1 pctx1
    Effect of <t>PcTX1</t> and small interfering RNA technology on the expression of ASIC1a and the activation of autophagy in rat articular chondrocytes. (A) Typical western blot image of the protein expression of ASIC1a. (B) Statistical results of the protein expression of ASIC1a. (C) Statistical results of the mRNA expression of ASIC1a. (D) Typical western blot image of the protein expression of LC3II. (E) Statistical results of the protein expression of LC3II. (F) Statistical results of the mRNA expression of Beclin1 and ULK1. (G) Articular chondrocytes were stained with acridine orange (magnification, ×200). Left panels, nuclei (stained green); middle panels, autophagolysosomes (stained orange-red); and right panels, merged. (H) Statistical results of acridine orange. Fluorescent microscopy demonstrated an increase in red fluorescence in acid - treated chondrocytes, indicating the presence of extracellular acidification as a marker of autophagy. ** P<0.01, vs. normal group; ## P<0.05, vs. pH 6.0 group. ASIC1a, acid-sensing ion channel 1a; PcTX1, <t>psalmotoxin-1;</t> LC3, microtubule-associated protein 1 light chain 3; ULK1, uncoordinated-51 like kinase 1; RNAi, RNA interference; NC, negative control.
    Psalmotoxin 1 Pctx1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Inhibition of acid - sensing ion channel 1a attenuates acid - induced activation of autophagy via a calcium signaling pathway in articular chondrocytes"

    Article Title: Inhibition of acid - sensing ion channel 1a attenuates acid - induced activation of autophagy via a calcium signaling pathway in articular chondrocytes

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2019.4085

    Effect of PcTX1 and small interfering RNA technology on the expression of ASIC1a and the activation of autophagy in rat articular chondrocytes. (A) Typical western blot image of the protein expression of ASIC1a. (B) Statistical results of the protein expression of ASIC1a. (C) Statistical results of the mRNA expression of ASIC1a. (D) Typical western blot image of the protein expression of LC3II. (E) Statistical results of the protein expression of LC3II. (F) Statistical results of the mRNA expression of Beclin1 and ULK1. (G) Articular chondrocytes were stained with acridine orange (magnification, ×200). Left panels, nuclei (stained green); middle panels, autophagolysosomes (stained orange-red); and right panels, merged. (H) Statistical results of acridine orange. Fluorescent microscopy demonstrated an increase in red fluorescence in acid - treated chondrocytes, indicating the presence of extracellular acidification as a marker of autophagy. ** P<0.01, vs. normal group; ## P<0.05, vs. pH 6.0 group. ASIC1a, acid-sensing ion channel 1a; PcTX1, psalmotoxin-1; LC3, microtubule-associated protein 1 light chain 3; ULK1, uncoordinated-51 like kinase 1; RNAi, RNA interference; NC, negative control.
    Figure Legend Snippet: Effect of PcTX1 and small interfering RNA technology on the expression of ASIC1a and the activation of autophagy in rat articular chondrocytes. (A) Typical western blot image of the protein expression of ASIC1a. (B) Statistical results of the protein expression of ASIC1a. (C) Statistical results of the mRNA expression of ASIC1a. (D) Typical western blot image of the protein expression of LC3II. (E) Statistical results of the protein expression of LC3II. (F) Statistical results of the mRNA expression of Beclin1 and ULK1. (G) Articular chondrocytes were stained with acridine orange (magnification, ×200). Left panels, nuclei (stained green); middle panels, autophagolysosomes (stained orange-red); and right panels, merged. (H) Statistical results of acridine orange. Fluorescent microscopy demonstrated an increase in red fluorescence in acid - treated chondrocytes, indicating the presence of extracellular acidification as a marker of autophagy. ** P<0.01, vs. normal group; ## P<0.05, vs. pH 6.0 group. ASIC1a, acid-sensing ion channel 1a; PcTX1, psalmotoxin-1; LC3, microtubule-associated protein 1 light chain 3; ULK1, uncoordinated-51 like kinase 1; RNAi, RNA interference; NC, negative control.

    Techniques Used: Small Interfering RNA, Expressing, Activation Assay, Western Blot, Staining, Microscopy, Fluorescence, Marker, Negative Control

    Knockdown of ASIC1a and PcTX1 reduces intracellular [Ca 2+ ] i in acid-mediated articular chondrocytes. (A) Cellular confocal micrographs showing changes in [Ca 2+ ] i concentration, as visualized by Fluo - 3 - AM in chondrocytes (magnification, ×200); (a) acid - induced elevation of [Ca 2+ ] i in Ca 2+ -free extracellular solution; (b) acid-induced elevation of [Ca 2+ ] i in extracellular Ca 2+ solution; (c) acid-induced elevation of [Ca 2+ ] i in chondrocytes treated with PcTX1; (d) acid-induced elevation of [Ca 2+ ] i in chondrocytes treated with ASIC1a - specific RNAi; (e) acid - induced elevation of [Ca 2+ ] i in chondrocytes treated with NC-RNAi. (B) Intracellular Ca 2+ imaging; (a) acid-induced elevation of [Ca 2+ ] i in Ca 2+ -free extracellular solution; (b) acid-induced elevation of [Ca 2+ ] i in extracellular Ca 2+ solution; (c) acid-induced elevation of [Ca 2+ ] i in chondrocytes treated with PcTX1; (d) acid-induced elevation of [Ca 2+ ] i in chondrocytes treated with ASIC1a - specific RNAi; (e) acid - induced elevation of [Ca 2+ ] i in chondrocytes treated with NC-RNAi. ASIC1a, acid-sensing ion channel 1a; PcTX1, psalmotoxin-1; RNAi, RNA interference; NC, negative control.
    Figure Legend Snippet: Knockdown of ASIC1a and PcTX1 reduces intracellular [Ca 2+ ] i in acid-mediated articular chondrocytes. (A) Cellular confocal micrographs showing changes in [Ca 2+ ] i concentration, as visualized by Fluo - 3 - AM in chondrocytes (magnification, ×200); (a) acid - induced elevation of [Ca 2+ ] i in Ca 2+ -free extracellular solution; (b) acid-induced elevation of [Ca 2+ ] i in extracellular Ca 2+ solution; (c) acid-induced elevation of [Ca 2+ ] i in chondrocytes treated with PcTX1; (d) acid-induced elevation of [Ca 2+ ] i in chondrocytes treated with ASIC1a - specific RNAi; (e) acid - induced elevation of [Ca 2+ ] i in chondrocytes treated with NC-RNAi. (B) Intracellular Ca 2+ imaging; (a) acid-induced elevation of [Ca 2+ ] i in Ca 2+ -free extracellular solution; (b) acid-induced elevation of [Ca 2+ ] i in extracellular Ca 2+ solution; (c) acid-induced elevation of [Ca 2+ ] i in chondrocytes treated with PcTX1; (d) acid-induced elevation of [Ca 2+ ] i in chondrocytes treated with ASIC1a - specific RNAi; (e) acid - induced elevation of [Ca 2+ ] i in chondrocytes treated with NC-RNAi. ASIC1a, acid-sensing ion channel 1a; PcTX1, psalmotoxin-1; RNAi, RNA interference; NC, negative control.

    Techniques Used: Concentration Assay, Imaging, Negative Control

    psalmotoxin 1 pctx1  (Alomone Labs)


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    Alomone Labs psalmotoxin 1 pctx1
    Psalmotoxin 1 Pctx1, supplied by Alomone Labs, 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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    psalmotoxin 1 pctx1  (Alomone Labs)


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    psalmotoxin 1 pctx1  (Alomone Labs)


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    Alomone Labs psalmotoxin 1 pctx1
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    psalmotoxin 1 pctx1  (Alomone Labs)


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    Alomone Labs psalmotoxin 1 pctx1
    ASIC-1as are activated by a decrease of extracellular pH in MNTB neuron. a, H+-gated IASICs from MNTB neurons in WT mice during transient acidification (4 s) of the extracellular media from pH 7.3 to 5.5 (mean peak amplitude, 2.5 ± 0.5 nA; n = 25), pH 6.0 (mean peak amplitude, 0.94 ± 0.22 nA; n = 20), or pH 6.5 (mean peak amplitude, 0.32 ± 0.04 nA; n = 6). MNTB neurons were whole-cell patch clamped at a holding potential of −70 mV. b, Acidification of the extracellular media induced no current in ASIC1a−/− mice. Please note the different magnitude in the current calibration bar. c, Activation of ASICs by a drop in pH from 7.3 to 6.0 induced membrane depolarization and triggered action potentials (shown in an expanded timescale in the inset at the right) in MNTB neurons from WT mice. d, In the presence of amiloride (150 μm), a nonspecific ASIC blocker, the amplitudes of IASICs triggered by dropping the pH from 7.4 to 6.0 were reduced in 82 ± 4% in WT mice (n = 12). The effect was reversible after washout. e, Effect of <t>PcTx1</t> (10 nm), a specific inhibitor of ASIC-1a homomers, on ASIC currents. The mean inhibition of IASIC peak value (n = 7) was 90 ± 3%.
    Psalmotoxin 1 Pctx1, supplied by Alomone Labs, 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 "Acid-Sensing Ion Channels Activated by Evoked Released Protons Modulate Synaptic Transmission at the Mouse Calyx of Held Synapse"

    Article Title: Acid-Sensing Ion Channels Activated by Evoked Released Protons Modulate Synaptic Transmission at the Mouse Calyx of Held Synapse

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.2566-16.2017

    ASIC-1as are activated by a decrease of extracellular pH in MNTB neuron. a, H+-gated IASICs from MNTB neurons in WT mice during transient acidification (4 s) of the extracellular media from pH 7.3 to 5.5 (mean peak amplitude, 2.5 ± 0.5 nA; n = 25), pH 6.0 (mean peak amplitude, 0.94 ± 0.22 nA; n = 20), or pH 6.5 (mean peak amplitude, 0.32 ± 0.04 nA; n = 6). MNTB neurons were whole-cell patch clamped at a holding potential of −70 mV. b, Acidification of the extracellular media induced no current in ASIC1a−/− mice. Please note the different magnitude in the current calibration bar. c, Activation of ASICs by a drop in pH from 7.3 to 6.0 induced membrane depolarization and triggered action potentials (shown in an expanded timescale in the inset at the right) in MNTB neurons from WT mice. d, In the presence of amiloride (150 μm), a nonspecific ASIC blocker, the amplitudes of IASICs triggered by dropping the pH from 7.4 to 6.0 were reduced in 82 ± 4% in WT mice (n = 12). The effect was reversible after washout. e, Effect of PcTx1 (10 nm), a specific inhibitor of ASIC-1a homomers, on ASIC currents. The mean inhibition of IASIC peak value (n = 7) was 90 ± 3%.
    Figure Legend Snippet: ASIC-1as are activated by a decrease of extracellular pH in MNTB neuron. a, H+-gated IASICs from MNTB neurons in WT mice during transient acidification (4 s) of the extracellular media from pH 7.3 to 5.5 (mean peak amplitude, 2.5 ± 0.5 nA; n = 25), pH 6.0 (mean peak amplitude, 0.94 ± 0.22 nA; n = 20), or pH 6.5 (mean peak amplitude, 0.32 ± 0.04 nA; n = 6). MNTB neurons were whole-cell patch clamped at a holding potential of −70 mV. b, Acidification of the extracellular media induced no current in ASIC1a−/− mice. Please note the different magnitude in the current calibration bar. c, Activation of ASICs by a drop in pH from 7.3 to 6.0 induced membrane depolarization and triggered action potentials (shown in an expanded timescale in the inset at the right) in MNTB neurons from WT mice. d, In the presence of amiloride (150 μm), a nonspecific ASIC blocker, the amplitudes of IASICs triggered by dropping the pH from 7.4 to 6.0 were reduced in 82 ± 4% in WT mice (n = 12). The effect was reversible after washout. e, Effect of PcTx1 (10 nm), a specific inhibitor of ASIC-1a homomers, on ASIC currents. The mean inhibition of IASIC peak value (n = 7) was 90 ± 3%.

    Techniques Used: Activation Assay, Inhibition

    Voltage dependence of ASIC currents and ASIC permeability to Ca2+. a, Representative traces showing IASICs activated by a 3-s-long pH drop from 7.3 to 6.5 while MNTB neurons were whole-cell patch clamped at different holding potentials. After returning to pH 7.3, cells were allowed to recover for 2 min. b, Mean I–V plot for ASIC currents activated by a 3-s-long pH drop from 7.3 to 6.5 (n = 6). The detection of calcium transients evoked during the activation of ASIC-1as by H+ injection. c, Representative traces of ASIC currents evoked by H+ iontophoresis in WT mice. The bar indicates the positive current pulse (2 nA, 3 s-duration) applied to a micropipette filled with HCl 0.1 m through a monopolar filament. d, Changes in calcium-sensitive indicator ΔF/F0 as a function of time. The arrow indicates the time of iontophoretic injection of H+. In MNTB neurons from WT mice, the Ca2+-dependent fluorescence rises up to 15 ± 3% (filled squares, n = 9), indicating that Ca2+ enters into the MNTB neuron through ASIC-1as when these are activated by H+. This increment in [Ca2+] is abolished when PcTx1 is applied to the bath solution (open squares; n = 8) and was not observed in MNTB neurons from ASIC1a−/− mice (filled triangles; n = 4).
    Figure Legend Snippet: Voltage dependence of ASIC currents and ASIC permeability to Ca2+. a, Representative traces showing IASICs activated by a 3-s-long pH drop from 7.3 to 6.5 while MNTB neurons were whole-cell patch clamped at different holding potentials. After returning to pH 7.3, cells were allowed to recover for 2 min. b, Mean I–V plot for ASIC currents activated by a 3-s-long pH drop from 7.3 to 6.5 (n = 6). The detection of calcium transients evoked during the activation of ASIC-1as by H+ injection. c, Representative traces of ASIC currents evoked by H+ iontophoresis in WT mice. The bar indicates the positive current pulse (2 nA, 3 s-duration) applied to a micropipette filled with HCl 0.1 m through a monopolar filament. d, Changes in calcium-sensitive indicator ΔF/F0 as a function of time. The arrow indicates the time of iontophoretic injection of H+. In MNTB neurons from WT mice, the Ca2+-dependent fluorescence rises up to 15 ± 3% (filled squares, n = 9), indicating that Ca2+ enters into the MNTB neuron through ASIC-1as when these are activated by H+. This increment in [Ca2+] is abolished when PcTx1 is applied to the bath solution (open squares; n = 8) and was not observed in MNTB neurons from ASIC1a−/− mice (filled triangles; n = 4).

    Techniques Used: Permeability, Activation Assay, Injection, Fluorescence

    Blockage of glutamatergic EPSCs reveals that protons released from presynaptic vesicles elicit postsynaptic ASIC-mediated currents and action potentials in MNTB neurons from WT mice. aI, Representative traces of EPSCs before (gray) and after (black) blocking AMPA, NMDA, GABA, and glycine receptors with CNQX (40 μm), d-APV (50 μm), bicuculline (20 μm), and strychnine (2 μm) in an MNTB neuron from a WT mouse [postnatal day 15 (P15)]. aII, Higher magnification of the ASIC-1a-mediated currents insensitive to CNQX, APV, bicuculline, and strychnine (black). Mean amplitudes were 46 ± 3 pA (n = 27). These ASIC-1a-mediated currents were highly reduced by amiloride (top, gray trace; n = 15) and were inhibited by PcTx1 (bottom, gray trace; n = 4). aIII, Top, Representative traces of EPSCs before (gray) and after (black) blocking AMPA, NMDA, GABA, and glycine receptors with CNQX (40 μm), d-APV (50 μm), bicuculline (20 μm), and strychnine (2 μm) in an MNTB neuron from ASIC1a−/− mouse (P16). Bottom, Higher-magnification image showing the absence of any current after postsynaptic receptor inhibition. b, Mean half-width and decay time of amiloride and PcTx1-sensitive ASIC-1a-mediated currents compared with glutamatergic EPSCs (HW: 1.31 ± 0.1 ms vs 0.59 ± 0.03 ms; DT: 3.0 ± 0.2 ms vs 1.18 ± 0.05 ms; n = 27; Student's t test, p < 0.001). c, Sample traces of ASIC-1a currents at different membrane potentials in WT mouse (P14). d, Increasing pH buffering using a 10 mm HEPES-based extracellular solution, pH 7.3, the ASIC-1a-mediated currents in MNTB neurons from WT mice were highly reduced (traces from a P15 mouse). e, ASIC-1a-mediated currents were highly diminished when the HEPES-based extracellular solution was maintained at pH 6.0 due to the desensitization of ASICs (traces from a P17 mouse). f, ASIC-1a-mediated currents (top, black trace) evoked by 100 Hz stimulation of presynaptic axons after the blockage of postsynaptic receptors with NBQX (a higher-affinity AMPA receptor antagonist, 10 μm) plus d-APV (50 μm), bicuculline (20 μm), and strychnine (2 μm) in a P15 WT mouse. Mean amplitudes were 38 ± 8 pA (HW, 1.1 ± 0.2 ms; DT, 2.7 ± 0.3 ms; n = 7). These currents were able to evoke APs (measured in current-clamp configuration at the resting potential of the MNTB neurons) whose amplitudes and kinetics did not differ from those evoked by glutamatergic currents (bottom, black trace; current-clamp recording). PcTx1 inhibited both ASIC-1a-mediated currents and evoked APs (gray traces).
    Figure Legend Snippet: Blockage of glutamatergic EPSCs reveals that protons released from presynaptic vesicles elicit postsynaptic ASIC-mediated currents and action potentials in MNTB neurons from WT mice. aI, Representative traces of EPSCs before (gray) and after (black) blocking AMPA, NMDA, GABA, and glycine receptors with CNQX (40 μm), d-APV (50 μm), bicuculline (20 μm), and strychnine (2 μm) in an MNTB neuron from a WT mouse [postnatal day 15 (P15)]. aII, Higher magnification of the ASIC-1a-mediated currents insensitive to CNQX, APV, bicuculline, and strychnine (black). Mean amplitudes were 46 ± 3 pA (n = 27). These ASIC-1a-mediated currents were highly reduced by amiloride (top, gray trace; n = 15) and were inhibited by PcTx1 (bottom, gray trace; n = 4). aIII, Top, Representative traces of EPSCs before (gray) and after (black) blocking AMPA, NMDA, GABA, and glycine receptors with CNQX (40 μm), d-APV (50 μm), bicuculline (20 μm), and strychnine (2 μm) in an MNTB neuron from ASIC1a−/− mouse (P16). Bottom, Higher-magnification image showing the absence of any current after postsynaptic receptor inhibition. b, Mean half-width and decay time of amiloride and PcTx1-sensitive ASIC-1a-mediated currents compared with glutamatergic EPSCs (HW: 1.31 ± 0.1 ms vs 0.59 ± 0.03 ms; DT: 3.0 ± 0.2 ms vs 1.18 ± 0.05 ms; n = 27; Student's t test, p < 0.001). c, Sample traces of ASIC-1a currents at different membrane potentials in WT mouse (P14). d, Increasing pH buffering using a 10 mm HEPES-based extracellular solution, pH 7.3, the ASIC-1a-mediated currents in MNTB neurons from WT mice were highly reduced (traces from a P15 mouse). e, ASIC-1a-mediated currents were highly diminished when the HEPES-based extracellular solution was maintained at pH 6.0 due to the desensitization of ASICs (traces from a P17 mouse). f, ASIC-1a-mediated currents (top, black trace) evoked by 100 Hz stimulation of presynaptic axons after the blockage of postsynaptic receptors with NBQX (a higher-affinity AMPA receptor antagonist, 10 μm) plus d-APV (50 μm), bicuculline (20 μm), and strychnine (2 μm) in a P15 WT mouse. Mean amplitudes were 38 ± 8 pA (HW, 1.1 ± 0.2 ms; DT, 2.7 ± 0.3 ms; n = 7). These currents were able to evoke APs (measured in current-clamp configuration at the resting potential of the MNTB neurons) whose amplitudes and kinetics did not differ from those evoked by glutamatergic currents (bottom, black trace; current-clamp recording). PcTx1 inhibited both ASIC-1a-mediated currents and evoked APs (gray traces).

    Techniques Used: Blocking Assay, Inhibition

    ASIC-1a-dependent increase of intracellular Ca2+ in MNTB neurons during HFS. a, Time course of the Fluo 8 fluorescence emission ratio (ΔF/F0) in postsynaptic MNTB neurons from WT mice (n = 9) during presynaptic HFS (150 Hz, 0.4 s; indicated by gray bar) in normal bicarbonate-based aCSF. Images were taken every 140 ms (first six images before stimulation served as the baseline). Left, During HFS, a maximum increase in intracellular Ca2+ of 28 ± 5% (n = 9) is observed (filled squares; control). After AMPA/kainite glutamate receptor inhibition with 40 μm CNQX (open squares, CNQX), a small component of the fluorescence increment (peak, 3.8 ± 0.9%; n = 9) remained during 150 Hz stimulation. PcTx1 inhibited this residual fluorescence (circles, + PcTx1; p < 0.05, one-way repeated-measure ANOVA). Right, Applying drugs in reverse order, PcTx1 reduced the maximum increase in intracellular Ca2+ of 29 ± 2% in control conditions (n = 10; filled squares, control) to 24 ± 3% (circles, PcTx1), while the addition of CNQX abolished any increase in fluorescence during 150 Hz stimulation (open squares, CNQX). b, Changing the pH buffer capacity of the aCSF alters ASIC-1a-dependent Ca2+ entry in MNTB neurons from WT mice. Plot of ΔF/F0 versus time during HFS in a low-pH buffer capacity aCSF before (filled squares, HEPES 1 mm; peak increase, 28 ± 8%; n = 9) and after the inhibition of glutamate AMPA and NMDA receptors (open squares, HEPES 1 mm plus CNQX+APV; peak increment, 3.2 ± 0.8%, n = 9). This remaining fluorescence component that may be attributed to Ca2+ influx through ASIC-1as was eliminated when a high-pH buffer capacity aCSF inhibits acidification (circles, HEPES 10 mm; n = 9; p < 0.05, one-way repeated-measures ANOVA). c, Time course of ΔF/F0 in postsynaptic MNTB neurons from ASIC1a−/− mice during HFS (150 Hz, 0.4 s) in normal bicarbonate-based aCSF (filled triangles, control; n = 8). The maximum increase in [Ca2+] through AMPA receptors (26 ± 5%) is not statistically different from that observed in WT mice (p > 0.05, Student's t test, WT vs ASIC-1a−/−). After glutamate AMPA and NMDA receptor inhibition, the increase in fluorescence is completely eliminated (open triangles, CNQX+APV; n = 8).
    Figure Legend Snippet: ASIC-1a-dependent increase of intracellular Ca2+ in MNTB neurons during HFS. a, Time course of the Fluo 8 fluorescence emission ratio (ΔF/F0) in postsynaptic MNTB neurons from WT mice (n = 9) during presynaptic HFS (150 Hz, 0.4 s; indicated by gray bar) in normal bicarbonate-based aCSF. Images were taken every 140 ms (first six images before stimulation served as the baseline). Left, During HFS, a maximum increase in intracellular Ca2+ of 28 ± 5% (n = 9) is observed (filled squares; control). After AMPA/kainite glutamate receptor inhibition with 40 μm CNQX (open squares, CNQX), a small component of the fluorescence increment (peak, 3.8 ± 0.9%; n = 9) remained during 150 Hz stimulation. PcTx1 inhibited this residual fluorescence (circles, + PcTx1; p < 0.05, one-way repeated-measure ANOVA). Right, Applying drugs in reverse order, PcTx1 reduced the maximum increase in intracellular Ca2+ of 29 ± 2% in control conditions (n = 10; filled squares, control) to 24 ± 3% (circles, PcTx1), while the addition of CNQX abolished any increase in fluorescence during 150 Hz stimulation (open squares, CNQX). b, Changing the pH buffer capacity of the aCSF alters ASIC-1a-dependent Ca2+ entry in MNTB neurons from WT mice. Plot of ΔF/F0 versus time during HFS in a low-pH buffer capacity aCSF before (filled squares, HEPES 1 mm; peak increase, 28 ± 8%; n = 9) and after the inhibition of glutamate AMPA and NMDA receptors (open squares, HEPES 1 mm plus CNQX+APV; peak increment, 3.2 ± 0.8%, n = 9). This remaining fluorescence component that may be attributed to Ca2+ influx through ASIC-1as was eliminated when a high-pH buffer capacity aCSF inhibits acidification (circles, HEPES 10 mm; n = 9; p < 0.05, one-way repeated-measures ANOVA). c, Time course of ΔF/F0 in postsynaptic MNTB neurons from ASIC1a−/− mice during HFS (150 Hz, 0.4 s) in normal bicarbonate-based aCSF (filled triangles, control; n = 8). The maximum increase in [Ca2+] through AMPA receptors (26 ± 5%) is not statistically different from that observed in WT mice (p > 0.05, Student's t test, WT vs ASIC-1a−/−). After glutamate AMPA and NMDA receptor inhibition, the increase in fluorescence is completely eliminated (open triangles, CNQX+APV; n = 8).

    Techniques Used: Fluorescence, Inhibition

    Enhanced STD at excitatory synaptic transmission in ASIC1a−/− mice compared with WT mice. a, Representative recording of AP-evoked EPSCs in WT and ASIC1a−/− MNTB neurons during 300 Hz stimulation. b, Time course of EPSC peak amplitudes (normalized to the first EPSC amplitude in the train) during 0.7 s stimulation at 300 Hz, fitted to a single exponential decay function, with a mean decay time constant τ = 7.0 ± 0.5 ms in WT mice (n = 42) and τ = 5.0 ± 0.6 ms in ASIC1a−/− mice (n = 21; p = 0.01, Student's t test). The EPSC amplitudes at the end of the stimuli reach a steady-state value of 9.3 ± 0.2% and 6.2 ± 0.2%, respectively, of the first EPSC amplitude in the train, in WT and ASIC1a−/− mice (p = 0.04, Student's t test). *p < 0.05, one-way repeated-measures ANOVA. c, Effect of PcTx1 on STD in WT mice. Normalized EPSC amplitudes as a function of time recorded in WT MNTB neurons during 300 Hz stimulation in bicarbonate-based aCSF at pH 7.3 before (filled squares) and during (open squares) the application of PcTx1 in the external solution. Data fitted to a single exponential decay function show an enhanced STD when ASIC-1as are blocked (mean decay time constants: before application of PcTx1, τ = 7.3 ± 0.6 ms; after application of PcTx1, τ = 5.96 ± 0.53 ms; n = 9). Inset, Individual and mean τ values, p = 4 × 10−5, paired student′s t test). Steady-state values of EPSC amplitudes at the end of the stimuli are also statistically different (before application of PcTx1, 9.4 ± 0.7% of the first EPSC amplitude in the train; after application of PcTx1, 8.8 ± 0.8% of the first EPSC amplitude in the train; p = 0.002, paired Student's t test). d, Effect of pH buffering on STD in WT mice. Time course of normalized EPSC amplitudes during 300 Hz stimulation recorded sequentially in a 1 mm (filled squares) and 10 mm (open squares) HEPES/MES-based aCSF (n = 8), fitted by single exponential decay functions. When acidification is inhibited by a high-pH buffer capacity solution, STD is increased (mean decay time constants: HEPES 1 mm: τ = 6.4 ± 0.7 ms; HEPES 10 mm: τ = 4.9 ± 0.6 ms). Inset, Individual τ values and mean (n = 8, p = 6 × 10−4, paired Student's t test). Steady-state values of EPSC amplitudes at the end of the stimuli are 9.5 ± 0.9% vs 8.3 ± 0.9% of the first EPSC amplitude in the train, in HEPES 1 mm and HEPES 10 mm, respectively (p = 0.005, paired Student's t test).
    Figure Legend Snippet: Enhanced STD at excitatory synaptic transmission in ASIC1a−/− mice compared with WT mice. a, Representative recording of AP-evoked EPSCs in WT and ASIC1a−/− MNTB neurons during 300 Hz stimulation. b, Time course of EPSC peak amplitudes (normalized to the first EPSC amplitude in the train) during 0.7 s stimulation at 300 Hz, fitted to a single exponential decay function, with a mean decay time constant τ = 7.0 ± 0.5 ms in WT mice (n = 42) and τ = 5.0 ± 0.6 ms in ASIC1a−/− mice (n = 21; p = 0.01, Student's t test). The EPSC amplitudes at the end of the stimuli reach a steady-state value of 9.3 ± 0.2% and 6.2 ± 0.2%, respectively, of the first EPSC amplitude in the train, in WT and ASIC1a−/− mice (p = 0.04, Student's t test). *p < 0.05, one-way repeated-measures ANOVA. c, Effect of PcTx1 on STD in WT mice. Normalized EPSC amplitudes as a function of time recorded in WT MNTB neurons during 300 Hz stimulation in bicarbonate-based aCSF at pH 7.3 before (filled squares) and during (open squares) the application of PcTx1 in the external solution. Data fitted to a single exponential decay function show an enhanced STD when ASIC-1as are blocked (mean decay time constants: before application of PcTx1, τ = 7.3 ± 0.6 ms; after application of PcTx1, τ = 5.96 ± 0.53 ms; n = 9). Inset, Individual and mean τ values, p = 4 × 10−5, paired student′s t test). Steady-state values of EPSC amplitudes at the end of the stimuli are also statistically different (before application of PcTx1, 9.4 ± 0.7% of the first EPSC amplitude in the train; after application of PcTx1, 8.8 ± 0.8% of the first EPSC amplitude in the train; p = 0.002, paired Student's t test). d, Effect of pH buffering on STD in WT mice. Time course of normalized EPSC amplitudes during 300 Hz stimulation recorded sequentially in a 1 mm (filled squares) and 10 mm (open squares) HEPES/MES-based aCSF (n = 8), fitted by single exponential decay functions. When acidification is inhibited by a high-pH buffer capacity solution, STD is increased (mean decay time constants: HEPES 1 mm: τ = 6.4 ± 0.7 ms; HEPES 10 mm: τ = 4.9 ± 0.6 ms). Inset, Individual τ values and mean (n = 8, p = 6 × 10−4, paired Student's t test). Steady-state values of EPSC amplitudes at the end of the stimuli are 9.5 ± 0.9% vs 8.3 ± 0.9% of the first EPSC amplitude in the train, in HEPES 1 mm and HEPES 10 mm, respectively (p = 0.005, paired Student's t test).

    Techniques Used: Transmission Assay

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    • pctx1  (Alomone Labs)
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    Alomone Labs pctx1
    Racemic lactate triggers ASIC1a dependent [Ca 2+ ] c and [Ca 2+ ] m signals. (A) Illustration of the figure hypothesis; (B) Representative Fura-2 ratio traces for [Ca 2+ ] c changes monitored in WT primary cultured hippocampal neurons (DIV 10–15) without and with pretreatment of the selective ASIC1a inhibitor <t>PcTx1</t> (20 nM, 120 s). Neurons were loaded with Fura-2AM (1 μM) and initially superfused with Ringer's solution at pH 7.4. Then, the superfusion was switched to Ringer's solution of pH 7.4 with an addition of DL-lactate (5 mM) as indicated by the arrowhead; (C) Quantification of the number of cytosolic (cyto) Ca 2+ peaks (transients) per 180-s time period measured as in (B) for WT neurons untreated (n = 39) and treated (n = 77); Box and whiskers plot show maximal and minimal values and all data points; (D) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in Rhod-2 AM (1 μM)-loaded WT neurons untreated and treated with PcTX1. Superfusion was switched to pH 7.4 Ringer's solution with DL-lactate; (E) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (D) for WT neurons untreated (n = 11) and treated with PcTX1 (n = 65); (F) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in response to direct puffing of the pH 7.4-DL- lactate solution in primary cultured WT and KO cortical neurons; (G) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (F) for WT (n = 6) and KO cortical neurons (n = 5); All summary data represent mean ± SD, ****p < 0.0001.
    Pctx1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    psalmotoxin 1 pctx1  (Alomone Labs)
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    Racemic lactate triggers ASIC1a dependent [Ca 2+ ] c and [Ca 2+ ] m signals. (A) Illustration of the figure hypothesis; (B) Representative Fura-2 ratio traces for [Ca 2+ ] c changes monitored in WT primary cultured hippocampal neurons (DIV 10–15) without and with pretreatment of the selective ASIC1a inhibitor <t>PcTx1</t> (20 nM, 120 s). Neurons were loaded with Fura-2AM (1 μM) and initially superfused with Ringer's solution at pH 7.4. Then, the superfusion was switched to Ringer's solution of pH 7.4 with an addition of DL-lactate (5 mM) as indicated by the arrowhead; (C) Quantification of the number of cytosolic (cyto) Ca 2+ peaks (transients) per 180-s time period measured as in (B) for WT neurons untreated (n = 39) and treated (n = 77); Box and whiskers plot show maximal and minimal values and all data points; (D) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in Rhod-2 AM (1 μM)-loaded WT neurons untreated and treated with PcTX1. Superfusion was switched to pH 7.4 Ringer's solution with DL-lactate; (E) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (D) for WT neurons untreated (n = 11) and treated with PcTX1 (n = 65); (F) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in response to direct puffing of the pH 7.4-DL- lactate solution in primary cultured WT and KO cortical neurons; (G) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (F) for WT (n = 6) and KO cortical neurons (n = 5); All summary data represent mean ± SD, ****p < 0.0001.
    Psalmotoxin 1 Pctx1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Racemic lactate triggers ASIC1a dependent [Ca 2+ ] c and [Ca 2+ ] m signals. (A) Illustration of the figure hypothesis; (B) Representative Fura-2 ratio traces for [Ca 2+ ] c changes monitored in WT primary cultured hippocampal neurons (DIV 10–15) without and with pretreatment of the selective ASIC1a inhibitor PcTx1 (20 nM, 120 s). Neurons were loaded with Fura-2AM (1 μM) and initially superfused with Ringer's solution at pH 7.4. Then, the superfusion was switched to Ringer's solution of pH 7.4 with an addition of DL-lactate (5 mM) as indicated by the arrowhead; (C) Quantification of the number of cytosolic (cyto) Ca 2+ peaks (transients) per 180-s time period measured as in (B) for WT neurons untreated (n = 39) and treated (n = 77); Box and whiskers plot show maximal and minimal values and all data points; (D) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in Rhod-2 AM (1 μM)-loaded WT neurons untreated and treated with PcTX1. Superfusion was switched to pH 7.4 Ringer's solution with DL-lactate; (E) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (D) for WT neurons untreated (n = 11) and treated with PcTX1 (n = 65); (F) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in response to direct puffing of the pH 7.4-DL- lactate solution in primary cultured WT and KO cortical neurons; (G) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (F) for WT (n = 6) and KO cortical neurons (n = 5); All summary data represent mean ± SD, ****p < 0.0001.

    Journal: Redox Biology

    Article Title: ASIC1a senses lactate uptake to regulate metabolism in neurons

    doi: 10.1016/j.redox.2022.102253

    Figure Lengend Snippet: Racemic lactate triggers ASIC1a dependent [Ca 2+ ] c and [Ca 2+ ] m signals. (A) Illustration of the figure hypothesis; (B) Representative Fura-2 ratio traces for [Ca 2+ ] c changes monitored in WT primary cultured hippocampal neurons (DIV 10–15) without and with pretreatment of the selective ASIC1a inhibitor PcTx1 (20 nM, 120 s). Neurons were loaded with Fura-2AM (1 μM) and initially superfused with Ringer's solution at pH 7.4. Then, the superfusion was switched to Ringer's solution of pH 7.4 with an addition of DL-lactate (5 mM) as indicated by the arrowhead; (C) Quantification of the number of cytosolic (cyto) Ca 2+ peaks (transients) per 180-s time period measured as in (B) for WT neurons untreated (n = 39) and treated (n = 77); Box and whiskers plot show maximal and minimal values and all data points; (D) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in Rhod-2 AM (1 μM)-loaded WT neurons untreated and treated with PcTX1. Superfusion was switched to pH 7.4 Ringer's solution with DL-lactate; (E) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (D) for WT neurons untreated (n = 11) and treated with PcTX1 (n = 65); (F) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in response to direct puffing of the pH 7.4-DL- lactate solution in primary cultured WT and KO cortical neurons; (G) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (F) for WT (n = 6) and KO cortical neurons (n = 5); All summary data represent mean ± SD, ****p < 0.0001.

    Article Snippet: Experiments done on WT neurons, untreated or treated with PcTX1 (Alomone labs, #STP200), were performed using the imaging system consisted of an Axiovert 100 inverted microscope (Zeiss), Polychrome V monochromator (TILL Photonics, Planegg, Germany) and a SensiCam cooled charge-coupled device (PCO, Kelheim, Germany).

    Techniques: Cell Culture, Fluorescence

    ASIC1a mediates L -lactate-induced increase in mitochondrial respiration and suppresses mitochondrial ROS production . Seahorse analysis (see Materials and Methods) was used to monitor mitochondrial respiration (OCR) with sequential additions of oligomycin (1 μM), FCCP (1 μM) + sodium pyruvate (5 mM), and a mix of rotenone/antimycin A (Ro/AA, 0.5 μM each), as indicated by the arrowheads, in media that contained or not D-, or L-lactate (5 mM); (A) Representative OCR plots of WT neurons in regular medium that contained no lactate (Ctrl) or the indicated lactate isomer and CIN4; (B) Quantification of maximal respiration as measured in (A) of WT neurons in regular medium (n = 6), and the medium that contained D- (n = 6) or L-lactate (n = 6), or L-lactate plus CIN4 (n = 6); (C) Representative OCR plots of KO neurons in regular medium or medium that contained the indicated lactate isomer and CIN4; (D) Quantification of maximal respiration as measured in (C) of KO in regular medium (Ctrl, n = 6) and media that contained the indicated D-lactate (n = 6), L-lactate (n-6), and L-lactate + CIN4 (n = 6); (E) Representative OCR plots of WT and KO neurons in L -lactate containing medium; (F) Quantification of maximal respiration as measured in (E) of WT (n = 6) and KO (n = 6) neurons; (G) Representative OCR plots of WT and KO neurons in L-lactate/CIN4 containing medium (n = 6); (H) Quantification of maximal respiration as measured in (G) of WT and KO neurons in L -lactate/CIN4 containing medium (n = 6 for each); (I) Representative RoGFP fluorescence traces for redox changes in response to L-lactate, H 2 O 2 and DTT added in the Ringer's solution in WT neurons untreated and treated with PcTX1; (J) Quantification of R/R 0 (480/405) at 500s after the addition of L-lactate (100s) as in (I) for WT neurons untreated (n = 9) and treated with PcTX1 (n = 7); (K) Schematic presentation of suggested pathway linking L-lactate to ASIC1a. All summary graph data represent mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

    Journal: Redox Biology

    Article Title: ASIC1a senses lactate uptake to regulate metabolism in neurons

    doi: 10.1016/j.redox.2022.102253

    Figure Lengend Snippet: ASIC1a mediates L -lactate-induced increase in mitochondrial respiration and suppresses mitochondrial ROS production . Seahorse analysis (see Materials and Methods) was used to monitor mitochondrial respiration (OCR) with sequential additions of oligomycin (1 μM), FCCP (1 μM) + sodium pyruvate (5 mM), and a mix of rotenone/antimycin A (Ro/AA, 0.5 μM each), as indicated by the arrowheads, in media that contained or not D-, or L-lactate (5 mM); (A) Representative OCR plots of WT neurons in regular medium that contained no lactate (Ctrl) or the indicated lactate isomer and CIN4; (B) Quantification of maximal respiration as measured in (A) of WT neurons in regular medium (n = 6), and the medium that contained D- (n = 6) or L-lactate (n = 6), or L-lactate plus CIN4 (n = 6); (C) Representative OCR plots of KO neurons in regular medium or medium that contained the indicated lactate isomer and CIN4; (D) Quantification of maximal respiration as measured in (C) of KO in regular medium (Ctrl, n = 6) and media that contained the indicated D-lactate (n = 6), L-lactate (n-6), and L-lactate + CIN4 (n = 6); (E) Representative OCR plots of WT and KO neurons in L -lactate containing medium; (F) Quantification of maximal respiration as measured in (E) of WT (n = 6) and KO (n = 6) neurons; (G) Representative OCR plots of WT and KO neurons in L-lactate/CIN4 containing medium (n = 6); (H) Quantification of maximal respiration as measured in (G) of WT and KO neurons in L -lactate/CIN4 containing medium (n = 6 for each); (I) Representative RoGFP fluorescence traces for redox changes in response to L-lactate, H 2 O 2 and DTT added in the Ringer's solution in WT neurons untreated and treated with PcTX1; (J) Quantification of R/R 0 (480/405) at 500s after the addition of L-lactate (100s) as in (I) for WT neurons untreated (n = 9) and treated with PcTX1 (n = 7); (K) Schematic presentation of suggested pathway linking L-lactate to ASIC1a. All summary graph data represent mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

    Article Snippet: Experiments done on WT neurons, untreated or treated with PcTX1 (Alomone labs, #STP200), were performed using the imaging system consisted of an Axiovert 100 inverted microscope (Zeiss), Polychrome V monochromator (TILL Photonics, Planegg, Germany) and a SensiCam cooled charge-coupled device (PCO, Kelheim, Germany).

    Techniques: Fluorescence