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mit-ateam 1.03 plasmid  (ATeam Scientific)

 
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    ATeam Scientific mit-ateam 1.03 plasmid
    Plasma-irradiated Cys products restore mitochondrial energy metabolism in cardiomyocytes under hypoxia. ( A ) Representative images of cardiomyocytes labeled with JC-1 dye to measure mitochondrial membrane potential. Cardiomyocytes treated with Cys or Cys plasma medium were cultured under normoxia or hypoxia. ΔCM(Cys)∗, plasma-irradiated methionine and cysteine-free DMEM supplemented with cysteine. Scale bars, 50 μm. Right: quantification of JC-1 red/green fluorescence ratio (n = 4 independent experiments). ( B ) Representative YFP/CFP ratiometric pseudocolored images of cardiomyocytes transfected with mit-ATeam 1.03 to monitor mitochondrial <t>ATP.</t> Scale bars, 50 μm. Right: quantification of <t>YFP/CFP</t> <t>FRET</t> ratio (n = 4 independent experiments). ( C ) NADH/NAD + ratio in cardiomyocyte lysates (n = 3 independent experiments). ( D ) Real-time oxygen consumption rate (OCR) measurements of cardiomyocytes cultured in plasma-irradiated Cys medium by Seahorse extracellular flux analyzer under normoxia. Cells were treated with oligomycin (Olig.), FCCP, rotenone (Rot.) and antimycin A (AA). ( E ) Measurements of OCR for different respiration modes (n = 3 independent experiments). ( F ) OCR measurements by MitoXpress Xtra assay. Cardiomyocytes were cultured under Nor or Hyp for 24 h, then basal respiration was measured under Nor (n = 3 independent experiments). ( G ) Lactate concentration in media was measured (n = 4 independent experiments). ( H ) Cardiomyocytes were labeled with JC-1 dye after gene silencing by siRNA transfection under Hyp, and JC-1 red/green fluorescence ratio was quantified. siNC: siRNA for negative control, siSqor: siRNA for Sqor gene (n = 3 independent experiments). Scale bars, 100 μm. ( I ) Cardiomyocytes transfected with EGFP and SQOR or empty vector (pcDNA) were labeled with TMRE to measure mitochondrial membrane potential. Right: Measurement of TMRE fluorescence intensity normalized by EGFP-positive cell surface area (n > 72 cells from 3 independent experiments). Scale bars, 50 μm. Data are shown as the means ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 by one-way ANOVA (A-C, F–H); unpaired t -test (E, I). “ns” indicates not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
    Mit Ateam 1.03 Plasmid, supplied by ATeam Scientific, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/mit-ateam+assay/pmc11663985-278-5-7?v=ATeam+Scientific
    Average 90 stars, based on 1 article reviews
    mit-ateam 1.03 plasmid - by Bioz Stars, 2026-07
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    Images

    1) Product Images from "Non-thermal atmospheric pressure plasma-irradiated cysteine protects cardiac ischemia/reperfusion injury by preserving supersulfides"

    Article Title: Non-thermal atmospheric pressure plasma-irradiated cysteine protects cardiac ischemia/reperfusion injury by preserving supersulfides

    Journal: Redox Biology

    doi: 10.1016/j.redox.2024.103445

    Plasma-irradiated Cys products restore mitochondrial energy metabolism in cardiomyocytes under hypoxia. ( A ) Representative images of cardiomyocytes labeled with JC-1 dye to measure mitochondrial membrane potential. Cardiomyocytes treated with Cys or Cys plasma medium were cultured under normoxia or hypoxia. ΔCM(Cys)∗, plasma-irradiated methionine and cysteine-free DMEM supplemented with cysteine. Scale bars, 50 μm. Right: quantification of JC-1 red/green fluorescence ratio (n = 4 independent experiments). ( B ) Representative YFP/CFP ratiometric pseudocolored images of cardiomyocytes transfected with mit-ATeam 1.03 to monitor mitochondrial ATP. Scale bars, 50 μm. Right: quantification of YFP/CFP FRET ratio (n = 4 independent experiments). ( C ) NADH/NAD + ratio in cardiomyocyte lysates (n = 3 independent experiments). ( D ) Real-time oxygen consumption rate (OCR) measurements of cardiomyocytes cultured in plasma-irradiated Cys medium by Seahorse extracellular flux analyzer under normoxia. Cells were treated with oligomycin (Olig.), FCCP, rotenone (Rot.) and antimycin A (AA). ( E ) Measurements of OCR for different respiration modes (n = 3 independent experiments). ( F ) OCR measurements by MitoXpress Xtra assay. Cardiomyocytes were cultured under Nor or Hyp for 24 h, then basal respiration was measured under Nor (n = 3 independent experiments). ( G ) Lactate concentration in media was measured (n = 4 independent experiments). ( H ) Cardiomyocytes were labeled with JC-1 dye after gene silencing by siRNA transfection under Hyp, and JC-1 red/green fluorescence ratio was quantified. siNC: siRNA for negative control, siSqor: siRNA for Sqor gene (n = 3 independent experiments). Scale bars, 100 μm. ( I ) Cardiomyocytes transfected with EGFP and SQOR or empty vector (pcDNA) were labeled with TMRE to measure mitochondrial membrane potential. Right: Measurement of TMRE fluorescence intensity normalized by EGFP-positive cell surface area (n > 72 cells from 3 independent experiments). Scale bars, 50 μm. Data are shown as the means ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 by one-way ANOVA (A-C, F–H); unpaired t -test (E, I). “ns” indicates not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
    Figure Legend Snippet: Plasma-irradiated Cys products restore mitochondrial energy metabolism in cardiomyocytes under hypoxia. ( A ) Representative images of cardiomyocytes labeled with JC-1 dye to measure mitochondrial membrane potential. Cardiomyocytes treated with Cys or Cys plasma medium were cultured under normoxia or hypoxia. ΔCM(Cys)∗, plasma-irradiated methionine and cysteine-free DMEM supplemented with cysteine. Scale bars, 50 μm. Right: quantification of JC-1 red/green fluorescence ratio (n = 4 independent experiments). ( B ) Representative YFP/CFP ratiometric pseudocolored images of cardiomyocytes transfected with mit-ATeam 1.03 to monitor mitochondrial ATP. Scale bars, 50 μm. Right: quantification of YFP/CFP FRET ratio (n = 4 independent experiments). ( C ) NADH/NAD + ratio in cardiomyocyte lysates (n = 3 independent experiments). ( D ) Real-time oxygen consumption rate (OCR) measurements of cardiomyocytes cultured in plasma-irradiated Cys medium by Seahorse extracellular flux analyzer under normoxia. Cells were treated with oligomycin (Olig.), FCCP, rotenone (Rot.) and antimycin A (AA). ( E ) Measurements of OCR for different respiration modes (n = 3 independent experiments). ( F ) OCR measurements by MitoXpress Xtra assay. Cardiomyocytes were cultured under Nor or Hyp for 24 h, then basal respiration was measured under Nor (n = 3 independent experiments). ( G ) Lactate concentration in media was measured (n = 4 independent experiments). ( H ) Cardiomyocytes were labeled with JC-1 dye after gene silencing by siRNA transfection under Hyp, and JC-1 red/green fluorescence ratio was quantified. siNC: siRNA for negative control, siSqor: siRNA for Sqor gene (n = 3 independent experiments). Scale bars, 100 μm. ( I ) Cardiomyocytes transfected with EGFP and SQOR or empty vector (pcDNA) were labeled with TMRE to measure mitochondrial membrane potential. Right: Measurement of TMRE fluorescence intensity normalized by EGFP-positive cell surface area (n > 72 cells from 3 independent experiments). Scale bars, 50 μm. Data are shown as the means ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 by one-way ANOVA (A-C, F–H); unpaired t -test (E, I). “ns” indicates not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

    Techniques Used: Clinical Proteomics, Irradiation, Labeling, Membrane, Cell Culture, Fluorescence, Transfection, Concentration Assay, Negative Control, Plasmid Preparation



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    Plasma-irradiated Cys products restore mitochondrial energy metabolism in cardiomyocytes under hypoxia. ( A ) Representative images of cardiomyocytes labeled with JC-1 dye to measure mitochondrial membrane potential. Cardiomyocytes treated with Cys or Cys plasma medium were cultured under normoxia or hypoxia. ΔCM(Cys)∗, plasma-irradiated methionine and cysteine-free DMEM supplemented with cysteine. Scale bars, 50 μm. Right: quantification of JC-1 red/green fluorescence ratio (n = 4 independent experiments). ( B ) Representative YFP/CFP ratiometric pseudocolored images of cardiomyocytes transfected with mit-ATeam 1.03 to monitor mitochondrial <t>ATP.</t> Scale bars, 50 μm. Right: quantification of <t>YFP/CFP</t> <t>FRET</t> ratio (n = 4 independent experiments). ( C ) NADH/NAD + ratio in cardiomyocyte lysates (n = 3 independent experiments). ( D ) Real-time oxygen consumption rate (OCR) measurements of cardiomyocytes cultured in plasma-irradiated Cys medium by Seahorse extracellular flux analyzer under normoxia. Cells were treated with oligomycin (Olig.), FCCP, rotenone (Rot.) and antimycin A (AA). ( E ) Measurements of OCR for different respiration modes (n = 3 independent experiments). ( F ) OCR measurements by MitoXpress Xtra assay. Cardiomyocytes were cultured under Nor or Hyp for 24 h, then basal respiration was measured under Nor (n = 3 independent experiments). ( G ) Lactate concentration in media was measured (n = 4 independent experiments). ( H ) Cardiomyocytes were labeled with JC-1 dye after gene silencing by siRNA transfection under Hyp, and JC-1 red/green fluorescence ratio was quantified. siNC: siRNA for negative control, siSqor: siRNA for Sqor gene (n = 3 independent experiments). Scale bars, 100 μm. ( I ) Cardiomyocytes transfected with EGFP and SQOR or empty vector (pcDNA) were labeled with TMRE to measure mitochondrial membrane potential. Right: Measurement of TMRE fluorescence intensity normalized by EGFP-positive cell surface area (n > 72 cells from 3 independent experiments). Scale bars, 50 μm. Data are shown as the means ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 by one-way ANOVA (A-C, F–H); unpaired t -test (E, I). “ns” indicates not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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    Plasma-irradiated Cys products restore mitochondrial energy metabolism in cardiomyocytes under hypoxia. ( A ) Representative images of cardiomyocytes labeled with JC-1 dye to measure mitochondrial membrane potential. Cardiomyocytes treated with Cys or Cys plasma medium were cultured under normoxia or hypoxia. ΔCM(Cys)∗, plasma-irradiated methionine and cysteine-free DMEM supplemented with cysteine. Scale bars, 50 μm. Right: quantification of JC-1 red/green fluorescence ratio (n = 4 independent experiments). ( B ) Representative YFP/CFP ratiometric pseudocolored images of cardiomyocytes transfected with mit-ATeam 1.03 to monitor mitochondrial <t>ATP.</t> Scale bars, 50 μm. Right: quantification of <t>YFP/CFP</t> <t>FRET</t> ratio (n = 4 independent experiments). ( C ) NADH/NAD + ratio in cardiomyocyte lysates (n = 3 independent experiments). ( D ) Real-time oxygen consumption rate (OCR) measurements of cardiomyocytes cultured in plasma-irradiated Cys medium by Seahorse extracellular flux analyzer under normoxia. Cells were treated with oligomycin (Olig.), FCCP, rotenone (Rot.) and antimycin A (AA). ( E ) Measurements of OCR for different respiration modes (n = 3 independent experiments). ( F ) OCR measurements by MitoXpress Xtra assay. Cardiomyocytes were cultured under Nor or Hyp for 24 h, then basal respiration was measured under Nor (n = 3 independent experiments). ( G ) Lactate concentration in media was measured (n = 4 independent experiments). ( H ) Cardiomyocytes were labeled with JC-1 dye after gene silencing by siRNA transfection under Hyp, and JC-1 red/green fluorescence ratio was quantified. siNC: siRNA for negative control, siSqor: siRNA for Sqor gene (n = 3 independent experiments). Scale bars, 100 μm. ( I ) Cardiomyocytes transfected with EGFP and SQOR or empty vector (pcDNA) were labeled with TMRE to measure mitochondrial membrane potential. Right: Measurement of TMRE fluorescence intensity normalized by EGFP-positive cell surface area (n > 72 cells from 3 independent experiments). Scale bars, 50 μm. Data are shown as the means ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 by one-way ANOVA (A-C, F–H); unpaired t -test (E, I). “ns” indicates not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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    Plasma-irradiated Cys products restore mitochondrial energy metabolism in cardiomyocytes under hypoxia. ( A ) Representative images of cardiomyocytes labeled with JC-1 dye to measure mitochondrial membrane potential. Cardiomyocytes treated with Cys or Cys plasma medium were cultured under normoxia or hypoxia. ΔCM(Cys)∗, plasma-irradiated methionine and cysteine-free DMEM supplemented with cysteine. Scale bars, 50 μm. Right: quantification of JC-1 red/green fluorescence ratio (n = 4 independent experiments). ( B ) Representative YFP/CFP ratiometric pseudocolored images of cardiomyocytes transfected with mit-ATeam 1.03 to monitor mitochondrial <t>ATP.</t> Scale bars, 50 μm. Right: quantification of <t>YFP/CFP</t> <t>FRET</t> ratio (n = 4 independent experiments). ( C ) NADH/NAD + ratio in cardiomyocyte lysates (n = 3 independent experiments). ( D ) Real-time oxygen consumption rate (OCR) measurements of cardiomyocytes cultured in plasma-irradiated Cys medium by Seahorse extracellular flux analyzer under normoxia. Cells were treated with oligomycin (Olig.), FCCP, rotenone (Rot.) and antimycin A (AA). ( E ) Measurements of OCR for different respiration modes (n = 3 independent experiments). ( F ) OCR measurements by MitoXpress Xtra assay. Cardiomyocytes were cultured under Nor or Hyp for 24 h, then basal respiration was measured under Nor (n = 3 independent experiments). ( G ) Lactate concentration in media was measured (n = 4 independent experiments). ( H ) Cardiomyocytes were labeled with JC-1 dye after gene silencing by siRNA transfection under Hyp, and JC-1 red/green fluorescence ratio was quantified. siNC: siRNA for negative control, siSqor: siRNA for Sqor gene (n = 3 independent experiments). Scale bars, 100 μm. ( I ) Cardiomyocytes transfected with EGFP and SQOR or empty vector (pcDNA) were labeled with TMRE to measure mitochondrial membrane potential. Right: Measurement of TMRE fluorescence intensity normalized by EGFP-positive cell surface area (n > 72 cells from 3 independent experiments). Scale bars, 50 μm. Data are shown as the means ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 by one-way ANOVA (A-C, F–H); unpaired t -test (E, I). “ns” indicates not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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    Plasma-irradiated Cys products restore mitochondrial energy metabolism in cardiomyocytes under hypoxia. ( A ) Representative images of cardiomyocytes labeled with JC-1 dye to measure mitochondrial membrane potential. Cardiomyocytes treated with Cys or Cys plasma medium were cultured under normoxia or hypoxia. ΔCM(Cys)∗, plasma-irradiated methionine and cysteine-free DMEM supplemented with cysteine. Scale bars, 50 μm. Right: quantification of JC-1 red/green fluorescence ratio (n = 4 independent experiments). ( B ) Representative YFP/CFP ratiometric pseudocolored images of cardiomyocytes transfected with mit-ATeam 1.03 to monitor mitochondrial <t>ATP.</t> Scale bars, 50 μm. Right: quantification of <t>YFP/CFP</t> <t>FRET</t> ratio (n = 4 independent experiments). ( C ) NADH/NAD + ratio in cardiomyocyte lysates (n = 3 independent experiments). ( D ) Real-time oxygen consumption rate (OCR) measurements of cardiomyocytes cultured in plasma-irradiated Cys medium by Seahorse extracellular flux analyzer under normoxia. Cells were treated with oligomycin (Olig.), FCCP, rotenone (Rot.) and antimycin A (AA). ( E ) Measurements of OCR for different respiration modes (n = 3 independent experiments). ( F ) OCR measurements by MitoXpress Xtra assay. Cardiomyocytes were cultured under Nor or Hyp for 24 h, then basal respiration was measured under Nor (n = 3 independent experiments). ( G ) Lactate concentration in media was measured (n = 4 independent experiments). ( H ) Cardiomyocytes were labeled with JC-1 dye after gene silencing by siRNA transfection under Hyp, and JC-1 red/green fluorescence ratio was quantified. siNC: siRNA for negative control, siSqor: siRNA for Sqor gene (n = 3 independent experiments). Scale bars, 100 μm. ( I ) Cardiomyocytes transfected with EGFP and SQOR or empty vector (pcDNA) were labeled with TMRE to measure mitochondrial membrane potential. Right: Measurement of TMRE fluorescence intensity normalized by EGFP-positive cell surface area (n > 72 cells from 3 independent experiments). Scale bars, 50 μm. Data are shown as the means ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 by one-way ANOVA (A-C, F–H); unpaired t -test (E, I). “ns” indicates not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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    Plasma-irradiated Cys products restore mitochondrial energy metabolism in cardiomyocytes under hypoxia. ( A ) Representative images of cardiomyocytes labeled with JC-1 dye to measure mitochondrial membrane potential. Cardiomyocytes treated with Cys or Cys plasma medium were cultured under normoxia or hypoxia. ΔCM(Cys)∗, plasma-irradiated methionine and cysteine-free DMEM supplemented with cysteine. Scale bars, 50 μm. Right: quantification of JC-1 red/green fluorescence ratio (n = 4 independent experiments). ( B ) Representative YFP/CFP ratiometric pseudocolored images of cardiomyocytes transfected with mit-ATeam 1.03 to monitor mitochondrial <t>ATP.</t> Scale bars, 50 μm. Right: quantification of <t>YFP/CFP</t> <t>FRET</t> ratio (n = 4 independent experiments). ( C ) NADH/NAD + ratio in cardiomyocyte lysates (n = 3 independent experiments). ( D ) Real-time oxygen consumption rate (OCR) measurements of cardiomyocytes cultured in plasma-irradiated Cys medium by Seahorse extracellular flux analyzer under normoxia. Cells were treated with oligomycin (Olig.), FCCP, rotenone (Rot.) and antimycin A (AA). ( E ) Measurements of OCR for different respiration modes (n = 3 independent experiments). ( F ) OCR measurements by MitoXpress Xtra assay. Cardiomyocytes were cultured under Nor or Hyp for 24 h, then basal respiration was measured under Nor (n = 3 independent experiments). ( G ) Lactate concentration in media was measured (n = 4 independent experiments). ( H ) Cardiomyocytes were labeled with JC-1 dye after gene silencing by siRNA transfection under Hyp, and JC-1 red/green fluorescence ratio was quantified. siNC: siRNA for negative control, siSqor: siRNA for Sqor gene (n = 3 independent experiments). Scale bars, 100 μm. ( I ) Cardiomyocytes transfected with EGFP and SQOR or empty vector (pcDNA) were labeled with TMRE to measure mitochondrial membrane potential. Right: Measurement of TMRE fluorescence intensity normalized by EGFP-positive cell surface area (n > 72 cells from 3 independent experiments). Scale bars, 50 μm. Data are shown as the means ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 by one-way ANOVA (A-C, F–H); unpaired t -test (E, I). “ns” indicates not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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    Plasma-irradiated Cys products restore mitochondrial energy metabolism in cardiomyocytes under hypoxia. ( A ) Representative images of cardiomyocytes labeled with JC-1 dye to measure mitochondrial membrane potential. Cardiomyocytes treated with Cys or Cys plasma medium were cultured under normoxia or hypoxia. ΔCM(Cys)∗, plasma-irradiated methionine and cysteine-free DMEM supplemented with cysteine. Scale bars, 50 μm. Right: quantification of JC-1 red/green fluorescence ratio (n = 4 independent experiments). ( B ) Representative YFP/CFP ratiometric pseudocolored images of cardiomyocytes transfected with mit-ATeam 1.03 to monitor mitochondrial <t>ATP.</t> Scale bars, 50 μm. Right: quantification of <t>YFP/CFP</t> <t>FRET</t> ratio (n = 4 independent experiments). ( C ) NADH/NAD + ratio in cardiomyocyte lysates (n = 3 independent experiments). ( D ) Real-time oxygen consumption rate (OCR) measurements of cardiomyocytes cultured in plasma-irradiated Cys medium by Seahorse extracellular flux analyzer under normoxia. Cells were treated with oligomycin (Olig.), FCCP, rotenone (Rot.) and antimycin A (AA). ( E ) Measurements of OCR for different respiration modes (n = 3 independent experiments). ( F ) OCR measurements by MitoXpress Xtra assay. Cardiomyocytes were cultured under Nor or Hyp for 24 h, then basal respiration was measured under Nor (n = 3 independent experiments). ( G ) Lactate concentration in media was measured (n = 4 independent experiments). ( H ) Cardiomyocytes were labeled with JC-1 dye after gene silencing by siRNA transfection under Hyp, and JC-1 red/green fluorescence ratio was quantified. siNC: siRNA for negative control, siSqor: siRNA for Sqor gene (n = 3 independent experiments). Scale bars, 100 μm. ( I ) Cardiomyocytes transfected with EGFP and SQOR or empty vector (pcDNA) were labeled with TMRE to measure mitochondrial membrane potential. Right: Measurement of TMRE fluorescence intensity normalized by EGFP-positive cell surface area (n > 72 cells from 3 independent experiments). Scale bars, 50 μm. Data are shown as the means ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 by one-way ANOVA (A-C, F–H); unpaired t -test (E, I). “ns” indicates not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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    Image Search Results


    Plasma-irradiated Cys products restore mitochondrial energy metabolism in cardiomyocytes under hypoxia. ( A ) Representative images of cardiomyocytes labeled with JC-1 dye to measure mitochondrial membrane potential. Cardiomyocytes treated with Cys or Cys plasma medium were cultured under normoxia or hypoxia. ΔCM(Cys)∗, plasma-irradiated methionine and cysteine-free DMEM supplemented with cysteine. Scale bars, 50 μm. Right: quantification of JC-1 red/green fluorescence ratio (n = 4 independent experiments). ( B ) Representative YFP/CFP ratiometric pseudocolored images of cardiomyocytes transfected with mit-ATeam 1.03 to monitor mitochondrial ATP. Scale bars, 50 μm. Right: quantification of YFP/CFP FRET ratio (n = 4 independent experiments). ( C ) NADH/NAD + ratio in cardiomyocyte lysates (n = 3 independent experiments). ( D ) Real-time oxygen consumption rate (OCR) measurements of cardiomyocytes cultured in plasma-irradiated Cys medium by Seahorse extracellular flux analyzer under normoxia. Cells were treated with oligomycin (Olig.), FCCP, rotenone (Rot.) and antimycin A (AA). ( E ) Measurements of OCR for different respiration modes (n = 3 independent experiments). ( F ) OCR measurements by MitoXpress Xtra assay. Cardiomyocytes were cultured under Nor or Hyp for 24 h, then basal respiration was measured under Nor (n = 3 independent experiments). ( G ) Lactate concentration in media was measured (n = 4 independent experiments). ( H ) Cardiomyocytes were labeled with JC-1 dye after gene silencing by siRNA transfection under Hyp, and JC-1 red/green fluorescence ratio was quantified. siNC: siRNA for negative control, siSqor: siRNA for Sqor gene (n = 3 independent experiments). Scale bars, 100 μm. ( I ) Cardiomyocytes transfected with EGFP and SQOR or empty vector (pcDNA) were labeled with TMRE to measure mitochondrial membrane potential. Right: Measurement of TMRE fluorescence intensity normalized by EGFP-positive cell surface area (n > 72 cells from 3 independent experiments). Scale bars, 50 μm. Data are shown as the means ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 by one-way ANOVA (A-C, F–H); unpaired t -test (E, I). “ns” indicates not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

    Journal: Redox Biology

    Article Title: Non-thermal atmospheric pressure plasma-irradiated cysteine protects cardiac ischemia/reperfusion injury by preserving supersulfides

    doi: 10.1016/j.redox.2024.103445

    Figure Lengend Snippet: Plasma-irradiated Cys products restore mitochondrial energy metabolism in cardiomyocytes under hypoxia. ( A ) Representative images of cardiomyocytes labeled with JC-1 dye to measure mitochondrial membrane potential. Cardiomyocytes treated with Cys or Cys plasma medium were cultured under normoxia or hypoxia. ΔCM(Cys)∗, plasma-irradiated methionine and cysteine-free DMEM supplemented with cysteine. Scale bars, 50 μm. Right: quantification of JC-1 red/green fluorescence ratio (n = 4 independent experiments). ( B ) Representative YFP/CFP ratiometric pseudocolored images of cardiomyocytes transfected with mit-ATeam 1.03 to monitor mitochondrial ATP. Scale bars, 50 μm. Right: quantification of YFP/CFP FRET ratio (n = 4 independent experiments). ( C ) NADH/NAD + ratio in cardiomyocyte lysates (n = 3 independent experiments). ( D ) Real-time oxygen consumption rate (OCR) measurements of cardiomyocytes cultured in plasma-irradiated Cys medium by Seahorse extracellular flux analyzer under normoxia. Cells were treated with oligomycin (Olig.), FCCP, rotenone (Rot.) and antimycin A (AA). ( E ) Measurements of OCR for different respiration modes (n = 3 independent experiments). ( F ) OCR measurements by MitoXpress Xtra assay. Cardiomyocytes were cultured under Nor or Hyp for 24 h, then basal respiration was measured under Nor (n = 3 independent experiments). ( G ) Lactate concentration in media was measured (n = 4 independent experiments). ( H ) Cardiomyocytes were labeled with JC-1 dye after gene silencing by siRNA transfection under Hyp, and JC-1 red/green fluorescence ratio was quantified. siNC: siRNA for negative control, siSqor: siRNA for Sqor gene (n = 3 independent experiments). Scale bars, 100 μm. ( I ) Cardiomyocytes transfected with EGFP and SQOR or empty vector (pcDNA) were labeled with TMRE to measure mitochondrial membrane potential. Right: Measurement of TMRE fluorescence intensity normalized by EGFP-positive cell surface area (n > 72 cells from 3 independent experiments). Scale bars, 50 μm. Data are shown as the means ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 by one-way ANOVA (A-C, F–H); unpaired t -test (E, I). “ns” indicates not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

    Article Snippet: For FRET-based measurement of mitochondrial ATP concentration, mit-ATeam 1.03 plasmid was transfected into cardiomyocytes [ ] by LipofectamineTM 3000 transfection reagent (Thermo Fisher Scientific).

    Techniques: Clinical Proteomics, Irradiation, Labeling, Membrane, Cell Culture, Fluorescence, Transfection, Concentration Assay, Negative Control, Plasmid Preparation