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    Alomone Labs pctx1
    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 <t>PcTX1;</t> (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
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    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

    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

    Article Snippet: 4.4 Fluorescence imaging 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

    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

    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

    Article Snippet: 4.4 Fluorescence imaging 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