jc 1 dye  (Thermo Fisher)


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    Name:
    JC 1 Dye Mitochondrial Membrane Potential Probe
    Description:
    JC 1 is a novel cationic carbocyanine dye that accumulates in mitochondria The dye exists as a monomer at low concentrations and yields green fluorescence similar to fluorescein At higher concentrations the dye forms J aggregates that exhibit a broad excitation spectrum and an emission maximum at 590 nm These characteristics make JC 1 a sensitive marker for mitochondrial membrane potential Another dye with similar characteristics is JC 9 D 22421
    Catalog Number:
    t3168
    Price:
    None
    Applications:
    Apoptosis|Cell Analysis|Cell Viability & Cytotoxicity|Mitochondria|Mitochondria Function|Mitochondrial Function|Cell Structure|Cell Viability, Proliferation & Function
    Category:
    Labeling Detection Products
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    Structured Review

    Thermo Fisher jc 1 dye
    Coculture screen to identify tumor–stroma interactions reveals divergent interactions between fibroblasts and TNBC cells Schematic of screen design. TNBC cell lines were labeled with <t>JC‐1,</t> grown in monoculture or in coculture with primary fibroblast cells, and treated with one of 42 anticancer drugs. JC‐1 fluorescence was monitored using a fluorescence plate reader at 8‐hour intervals for 72 h. Representative images of BT‐20 cells (BL subtype) cocultured with HADF fibroblasts. BT‐20 cells labeled with JC‐1 dye; HADF labeled with a blue cell dye (CellTrace). Images taken before drug addition (B) or 96 h after exposure to 0.5 μM camptothecin (C). Kinetic trace of JC‐1 red fluorescence following exposure to camptothecin as in (B and C). Data are relative JC‐1 red fluorescence, normalized to the well's average prior to drug addition. Data represent mean ± standard deviation for five biological replicates. Total coculture screen data. Each blue dot represents a unique TNBC–fibroblast–drug measurement. 312,120 total measurements of drug response. Orange and purple dots highlight conditions validated in panels (F–I). Orange are HCC‐1143 (BL) cells cocultured with HCPF and treated with palbociclib. Purple dots are Hs578T (ML) cocultured with WS1 and exposed to etoposide. For colored dots, increasing size represents longer drug exposure times. Validation of coculture screening data. (F) Flow cytometry analysis of cell death using Live/Dead Blue stain. FarRed CellTrace was used to label and distinguish fibroblasts. (G) Quantification of percent TNBC cell death in experiment described in panel (F). (H and I) Flow cytometry and quantitative analysis, as in panels (F and G) for Hs578T +/− WS1 and treated with etoposide. Error bars represent standard deviation of biological replicates. P ‐value calculated using t ‐test. Data are from three biological replicates and error bars. *** P ‐value
    JC 1 is a novel cationic carbocyanine dye that accumulates in mitochondria The dye exists as a monomer at low concentrations and yields green fluorescence similar to fluorescein At higher concentrations the dye forms J aggregates that exhibit a broad excitation spectrum and an emission maximum at 590 nm These characteristics make JC 1 a sensitive marker for mitochondrial membrane potential Another dye with similar characteristics is JC 9 D 22421
    https://www.bioz.com/result/jc 1 dye/product/Thermo Fisher
    Average 99 stars, based on 171 article reviews
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    jc 1 dye - by Bioz Stars, 2021-01
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    Images

    1) Product Images from "Tumor‐stroma interactions differentially alter drug sensitivity based on the origin of stromal cells"

    Article Title: Tumor‐stroma interactions differentially alter drug sensitivity based on the origin of stromal cells

    Journal: Molecular Systems Biology

    doi: 10.15252/msb.20188322

    Coculture screen to identify tumor–stroma interactions reveals divergent interactions between fibroblasts and TNBC cells Schematic of screen design. TNBC cell lines were labeled with JC‐1, grown in monoculture or in coculture with primary fibroblast cells, and treated with one of 42 anticancer drugs. JC‐1 fluorescence was monitored using a fluorescence plate reader at 8‐hour intervals for 72 h. Representative images of BT‐20 cells (BL subtype) cocultured with HADF fibroblasts. BT‐20 cells labeled with JC‐1 dye; HADF labeled with a blue cell dye (CellTrace). Images taken before drug addition (B) or 96 h after exposure to 0.5 μM camptothecin (C). Kinetic trace of JC‐1 red fluorescence following exposure to camptothecin as in (B and C). Data are relative JC‐1 red fluorescence, normalized to the well's average prior to drug addition. Data represent mean ± standard deviation for five biological replicates. Total coculture screen data. Each blue dot represents a unique TNBC–fibroblast–drug measurement. 312,120 total measurements of drug response. Orange and purple dots highlight conditions validated in panels (F–I). Orange are HCC‐1143 (BL) cells cocultured with HCPF and treated with palbociclib. Purple dots are Hs578T (ML) cocultured with WS1 and exposed to etoposide. For colored dots, increasing size represents longer drug exposure times. Validation of coculture screening data. (F) Flow cytometry analysis of cell death using Live/Dead Blue stain. FarRed CellTrace was used to label and distinguish fibroblasts. (G) Quantification of percent TNBC cell death in experiment described in panel (F). (H and I) Flow cytometry and quantitative analysis, as in panels (F and G) for Hs578T +/− WS1 and treated with etoposide. Error bars represent standard deviation of biological replicates. P ‐value calculated using t ‐test. Data are from three biological replicates and error bars. *** P ‐value
    Figure Legend Snippet: Coculture screen to identify tumor–stroma interactions reveals divergent interactions between fibroblasts and TNBC cells Schematic of screen design. TNBC cell lines were labeled with JC‐1, grown in monoculture or in coculture with primary fibroblast cells, and treated with one of 42 anticancer drugs. JC‐1 fluorescence was monitored using a fluorescence plate reader at 8‐hour intervals for 72 h. Representative images of BT‐20 cells (BL subtype) cocultured with HADF fibroblasts. BT‐20 cells labeled with JC‐1 dye; HADF labeled with a blue cell dye (CellTrace). Images taken before drug addition (B) or 96 h after exposure to 0.5 μM camptothecin (C). Kinetic trace of JC‐1 red fluorescence following exposure to camptothecin as in (B and C). Data are relative JC‐1 red fluorescence, normalized to the well's average prior to drug addition. Data represent mean ± standard deviation for five biological replicates. Total coculture screen data. Each blue dot represents a unique TNBC–fibroblast–drug measurement. 312,120 total measurements of drug response. Orange and purple dots highlight conditions validated in panels (F–I). Orange are HCC‐1143 (BL) cells cocultured with HCPF and treated with palbociclib. Purple dots are Hs578T (ML) cocultured with WS1 and exposed to etoposide. For colored dots, increasing size represents longer drug exposure times. Validation of coculture screening data. (F) Flow cytometry analysis of cell death using Live/Dead Blue stain. FarRed CellTrace was used to label and distinguish fibroblasts. (G) Quantification of percent TNBC cell death in experiment described in panel (F). (H and I) Flow cytometry and quantitative analysis, as in panels (F and G) for Hs578T +/− WS1 and treated with etoposide. Error bars represent standard deviation of biological replicates. P ‐value calculated using t ‐test. Data are from three biological replicates and error bars. *** P ‐value

    Techniques Used: Labeling, Fluorescence, Standard Deviation, Flow Cytometry, Cytometry, Staining

    2) Product Images from "Cumulative mitochondrial activity correlates with ototoxin susceptibility in zebrafish mechanosensory hair cells"

    Article Title: Cumulative mitochondrial activity correlates with ototoxin susceptibility in zebrafish mechanosensory hair cells

    Journal: eLife

    doi: 10.7554/eLife.38062

    Mitochondrial polarization does not shift with short-term hair cell stimulation. Mean JC-1 fluorescence ratio measured following 90 min of orbital stimulation. Larvae were incubated in JC-1 just prior to orbital shaking. Control: 0.58 ± 0.17; Orbital Shaker: 0.57 ± 0.08; mean ratio ± SD, n = 10 fish per group, 2–3 neuromasts per fish; Mann-Whitney U test, p = 0.91).
    Figure Legend Snippet: Mitochondrial polarization does not shift with short-term hair cell stimulation. Mean JC-1 fluorescence ratio measured following 90 min of orbital stimulation. Larvae were incubated in JC-1 just prior to orbital shaking. Control: 0.58 ± 0.17; Orbital Shaker: 0.57 ± 0.08; mean ratio ± SD, n = 10 fish per group, 2–3 neuromasts per fish; Mann-Whitney U test, p = 0.91).

    Techniques Used: Cell Stimulation, Fluorescence, Incubation, Fluorescence In Situ Hybridization, MANN-WHITNEY

    Acute mitochondrial activity is reduced in the absence of MET. ( A, B ) Maximum projections of hair cells from WT/Het and sputnik mutant siblings incubated in JC-1 dye. Hair cells were imaged from a dorsal view, as indicated in the schematic shown in Figure 1—figure supplement 1B . ( C ) Mean JC-1 fluorescence plotted as a ratio of red:green. WT/Het: 0.25 ± 0.24 n = 8 fish; Mutant: 0.05 ± 0.07 n = 8 fish; mean ratio ± SD. Mann-Whitney U test was used to assess significance. Value for each fish represents the mean of 3 neuromasts. Scale bar = 5 μm.
    Figure Legend Snippet: Acute mitochondrial activity is reduced in the absence of MET. ( A, B ) Maximum projections of hair cells from WT/Het and sputnik mutant siblings incubated in JC-1 dye. Hair cells were imaged from a dorsal view, as indicated in the schematic shown in Figure 1—figure supplement 1B . ( C ) Mean JC-1 fluorescence plotted as a ratio of red:green. WT/Het: 0.25 ± 0.24 n = 8 fish; Mutant: 0.05 ± 0.07 n = 8 fish; mean ratio ± SD. Mann-Whitney U test was used to assess significance. Value for each fish represents the mean of 3 neuromasts. Scale bar = 5 μm.

    Techniques Used: Activity Assay, Mutagenesis, Incubation, Fluorescence, Fluorescence In Situ Hybridization, MANN-WHITNEY

    3) Product Images from "Evaluation of cationic channel TRPV2 as a novel biomarker and therapeutic target in Leukemia-Implications concerning the resolution of pulmonary inflammation"

    Article Title: Evaluation of cationic channel TRPV2 as a novel biomarker and therapeutic target in Leukemia-Implications concerning the resolution of pulmonary inflammation

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-37469-8

    TL and SKF activated the mitochondrial caspase-mediated apoptotic pathway. ( A ) dose-dependent collapse of MMP in K562 and U937 cells treated with TL or SKF for 48 h. Cells were stained with the lipophilic cationic dye JC-1 and analyzed by flow Cytometry. ( B ) K562 were treated for 48 h with TL or SKF at the indicated concentrations, then immuno-stained and analyzed by flow cytometry to detect active (cleaved) caspase-3 and cleaved PARP levels. ( C ) Assessment of DNA damage resulting from apoptotic cell death by flow cytometry analysis of active H2AX (γ-H2AX) levels in SKF- and TL-treated K562 cells. ( D ) Whole cell lysates of TL- and SKF-treated K562, U937, or THP-1 were processed for Western blot analysis to detect PARP, cleaved PARP, caspase-3, cleaved caspase-3, and anti-apoptotic marker Bcl2. ( E ) Immunoblotting analysis of whole cell extracts to detect changes in p38, phospho (p)-38, ERK1/2, and phospho (p)-ERK1/2 in K562 and U937 cells treated with TL or SKF for 48 h. Note that p38 and ERK1/2 were activated and inactivated respectively by both drugs. Data displayed in the graphs represent the mean ± SD. Refer to Supplementary Fig. 5S for more details.
    Figure Legend Snippet: TL and SKF activated the mitochondrial caspase-mediated apoptotic pathway. ( A ) dose-dependent collapse of MMP in K562 and U937 cells treated with TL or SKF for 48 h. Cells were stained with the lipophilic cationic dye JC-1 and analyzed by flow Cytometry. ( B ) K562 were treated for 48 h with TL or SKF at the indicated concentrations, then immuno-stained and analyzed by flow cytometry to detect active (cleaved) caspase-3 and cleaved PARP levels. ( C ) Assessment of DNA damage resulting from apoptotic cell death by flow cytometry analysis of active H2AX (γ-H2AX) levels in SKF- and TL-treated K562 cells. ( D ) Whole cell lysates of TL- and SKF-treated K562, U937, or THP-1 were processed for Western blot analysis to detect PARP, cleaved PARP, caspase-3, cleaved caspase-3, and anti-apoptotic marker Bcl2. ( E ) Immunoblotting analysis of whole cell extracts to detect changes in p38, phospho (p)-38, ERK1/2, and phospho (p)-ERK1/2 in K562 and U937 cells treated with TL or SKF for 48 h. Note that p38 and ERK1/2 were activated and inactivated respectively by both drugs. Data displayed in the graphs represent the mean ± SD. Refer to Supplementary Fig. 5S for more details.

    Techniques Used: Staining, Flow Cytometry, Cytometry, Western Blot, Marker

    4) Product Images from "Combination of Sulindac and Dichloroacetate Kills Cancer Cells via Oxidative Damage"

    Article Title: Combination of Sulindac and Dichloroacetate Kills Cancer Cells via Oxidative Damage

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039949

    The combination of sulindac and DCA causes disruption of the mitochondrial membrane potential in cancer cells. Top panels (A) illustrate the results for A549 cancer cells while the bottom panels (B) depict the results for SCC25 cancer cells. Mitochondrial membrane potential loss was detected by a change in JC-1 distribution resulting in an increase in green fluorescence (see Methods). The experimental conditions for JC-1 staining and fluorescent microscopy are explained in detail under Methods and the drug treatment regimens are depicted below the panels. Untreated cells (sub-panels A1 and B1), cells treated with sulindac (sub-panels A2 and B2), cells treated with DCA (sub-panels A3 and B3), and cells treated with sulindac and DCA (sub-panels A4 and B4). Several independent fields were photomicrographed and representative fields for each condition are shown.
    Figure Legend Snippet: The combination of sulindac and DCA causes disruption of the mitochondrial membrane potential in cancer cells. Top panels (A) illustrate the results for A549 cancer cells while the bottom panels (B) depict the results for SCC25 cancer cells. Mitochondrial membrane potential loss was detected by a change in JC-1 distribution resulting in an increase in green fluorescence (see Methods). The experimental conditions for JC-1 staining and fluorescent microscopy are explained in detail under Methods and the drug treatment regimens are depicted below the panels. Untreated cells (sub-panels A1 and B1), cells treated with sulindac (sub-panels A2 and B2), cells treated with DCA (sub-panels A3 and B3), and cells treated with sulindac and DCA (sub-panels A4 and B4). Several independent fields were photomicrographed and representative fields for each condition are shown.

    Techniques Used: Fluorescence, Staining, Microscopy

    5) Product Images from "Topical Application of the Antimicrobial Agent Triclosan Induces NLRP3 Inflammasome Activation and Mitochondrial Dysfunction"

    Article Title: Topical Application of the Antimicrobial Agent Triclosan Induces NLRP3 Inflammasome Activation and Mitochondrial Dysfunction

    Journal: Toxicological sciences : an official journal of the Society of Toxicology

    doi: 10.1093/toxsci/kfaa056

    Triclosan alters mitochondrial components in skin draining lymph nodes (dLNs). Mitochondrial ROS (mtROS) (A), mitochondrial mass (mtMass) (B), and mitochondrial membrane potential (MMP) (C) were measured in lymphocytes from dLNs after dermal exposure to triclosan via 5μM MitoSOX Red, 200nM MitoTracker Green FM, and 2μM JC-1, respectively. Using flow cytometry, median fluorescence intensity (MFI) was measured and normalized to the days corresponding 0% vehicle control (VC) (A, B). JC-1 dye undergoes a spectral shift upon depolarization and was analyzed via a red/green fluorescence intensity ratio (C). Data shown as the mean (± SEM) of 5 mice per group. Statistical significance relative to the days corresponding 0% VC determined by one-way ANOVA followed by Dunnett’s post-test indicated as * p
    Figure Legend Snippet: Triclosan alters mitochondrial components in skin draining lymph nodes (dLNs). Mitochondrial ROS (mtROS) (A), mitochondrial mass (mtMass) (B), and mitochondrial membrane potential (MMP) (C) were measured in lymphocytes from dLNs after dermal exposure to triclosan via 5μM MitoSOX Red, 200nM MitoTracker Green FM, and 2μM JC-1, respectively. Using flow cytometry, median fluorescence intensity (MFI) was measured and normalized to the days corresponding 0% vehicle control (VC) (A, B). JC-1 dye undergoes a spectral shift upon depolarization and was analyzed via a red/green fluorescence intensity ratio (C). Data shown as the mean (± SEM) of 5 mice per group. Statistical significance relative to the days corresponding 0% VC determined by one-way ANOVA followed by Dunnett’s post-test indicated as * p

    Techniques Used: Flow Cytometry, Fluorescence, Mouse Assay

    6) Product Images from "Synthesis and cytotoxicity studies of 1-propenyl-1,3-dihydro-benzimidazol-2-one"

    Article Title: Synthesis and cytotoxicity studies of 1-propenyl-1,3-dihydro-benzimidazol-2-one

    Journal: Journal of Chemical Biology

    doi: 10.1007/s12154-015-0130-8

    HEK 293 cells were treated with benzimidazolone compound for overnight then stained with JC-1 dye for checking mitochondrial health. The cells were then studied and imaged under a fluorescence microscope. First and second rows , respectively, show control
    Figure Legend Snippet: HEK 293 cells were treated with benzimidazolone compound for overnight then stained with JC-1 dye for checking mitochondrial health. The cells were then studied and imaged under a fluorescence microscope. First and second rows , respectively, show control

    Techniques Used: Staining, Fluorescence, Microscopy

    7) Product Images from "Cabbage (Brassica oleracea var. capitata) Protects against H2O2-Induced Oxidative Stress by Preventing Mitochondrial Dysfunction in H9c2 Cardiomyoblasts"

    Article Title: Cabbage (Brassica oleracea var. capitata) Protects against H2O2-Induced Oxidative Stress by Preventing Mitochondrial Dysfunction in H9c2 Cardiomyoblasts

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2018/2179021

    Cabbage extract prevents mitochondrial dysfunction upon H 2 O 2 -induced oxidative stress in H9c2 cardiomyocytes. The MMP was determined by incubating cells with JC-1 for 20 min. (a) Representative images of and (b) fluorescence intensities in H9c2 cardiomyocytes treated with 500 μ M H 2 O 2 for 24 h following pretreatment with 100, 200, and 300 μ g/ml cabbage extract for 24 h. (c) Quantitative RT-PCR analysis of mitochondrial biogenesis-related genes (NRF-1, PPAR α , ERR α , and PGC-1 β ). Western blot analysis was performed in triplicate with three independent samples. Data are expressed as fold changes ± SEM versus control cells. Significance was analyzed by a one-way ANOVA followed by the Bonferroni post hoc test. ## P
    Figure Legend Snippet: Cabbage extract prevents mitochondrial dysfunction upon H 2 O 2 -induced oxidative stress in H9c2 cardiomyocytes. The MMP was determined by incubating cells with JC-1 for 20 min. (a) Representative images of and (b) fluorescence intensities in H9c2 cardiomyocytes treated with 500 μ M H 2 O 2 for 24 h following pretreatment with 100, 200, and 300 μ g/ml cabbage extract for 24 h. (c) Quantitative RT-PCR analysis of mitochondrial biogenesis-related genes (NRF-1, PPAR α , ERR α , and PGC-1 β ). Western blot analysis was performed in triplicate with three independent samples. Data are expressed as fold changes ± SEM versus control cells. Significance was analyzed by a one-way ANOVA followed by the Bonferroni post hoc test. ## P

    Techniques Used: Fluorescence, Quantitative RT-PCR, Pyrolysis Gas Chromatography, Western Blot

    8) Product Images from "Glucose-6-phosphate dehydrogenase is critical for suppression of cardiac hypertrophy by H2S"

    Article Title: Glucose-6-phosphate dehydrogenase is critical for suppression of cardiac hypertrophy by H2S

    Journal: Cell Death Discovery

    doi: 10.1038/s41420-017-0010-9

    β-adrenergic stimulation downregulates endogenous H 2 S levels a – d The cells were stained with H 2 S-specific dye, SF7-AM (2.5 µM) and relative levels of endogenous H 2 S estimated in cardiac cells, with or without β-AR stimulation (employing ISO/NE), in the presence or absence of NaHS pre-treatment (400 µM, 30 min). The representative fluorescence micrographs (10X magnification, λ ex/em : 495/520 nm) for specific groups ( n = 3) are shown in panels a and c . Magnified regions (inset) and scale bar is also shown for images in all groups. Panels b and d depict box-whisker plots representing corrected total cell fluorescence, CTCF, for indicated groups. The relative change in mean CTCF (represented as % of control) is also indicated in parenthesis. e–h The cells were treated with NaHS (400 µM) or H 2 O 2 (50 µM) for indicated duration of time and specific assays were performed. e Mitochondrial superoxide measurement utilizing Mitosox red (mitochondrial superoxide-specific probe) staining and flow cytometry (BD Accuri C6) f Mitochondrial membrane potential (ΔΨm) assessment utilizing JC-1 dye and flow cytometry (BD Accuri C6). FL-1 (green) vs. FL-2 (red) dot plots were generated for the indicated groups. Cells with normal ΔΨm are detected as those with higher FL-2 signal (FL-1 bright , FL-2 bright cells)—indicative of higher levels of JC-1 aggregates (detected in FL-2, 570 nm). The loss of mitochondrial membrane potential results in increased levels of FL-1 bright , FL-2 dim cells—indicative of higher JC-1 monomer (detected in FL-1, 530 nm). g Apoptosis assay: the cells were subjected to Annexin V-FITC/Propidium Iodide (PI) double staining and data acquired using flow cytometry (BD FACS Calibur). FITC − /PI − cells indicate normal cells while FITC + /PI − , FITC + /PI + and PI + suggest the presence of cells in specific stages of apoptosis (early, late, or necrotic). The percentage of cells in individual quadrant is also indicated in the figure. h DNA content analysis: the cells were stained with propidium iodide and DNA content analysis (cell cycle) done employing flow cytometry (BD FACS Calibur). Histogram plot for specific groups is as shown. The relative fractions of cells in specific stages of cell cycle (G1, S or G2) are also indicated. H 2 O 2 treated group also showed distinctive sub-G1 population, indicating the presence of apoptotic cells.
    Figure Legend Snippet: β-adrenergic stimulation downregulates endogenous H 2 S levels a – d The cells were stained with H 2 S-specific dye, SF7-AM (2.5 µM) and relative levels of endogenous H 2 S estimated in cardiac cells, with or without β-AR stimulation (employing ISO/NE), in the presence or absence of NaHS pre-treatment (400 µM, 30 min). The representative fluorescence micrographs (10X magnification, λ ex/em : 495/520 nm) for specific groups ( n = 3) are shown in panels a and c . Magnified regions (inset) and scale bar is also shown for images in all groups. Panels b and d depict box-whisker plots representing corrected total cell fluorescence, CTCF, for indicated groups. The relative change in mean CTCF (represented as % of control) is also indicated in parenthesis. e–h The cells were treated with NaHS (400 µM) or H 2 O 2 (50 µM) for indicated duration of time and specific assays were performed. e Mitochondrial superoxide measurement utilizing Mitosox red (mitochondrial superoxide-specific probe) staining and flow cytometry (BD Accuri C6) f Mitochondrial membrane potential (ΔΨm) assessment utilizing JC-1 dye and flow cytometry (BD Accuri C6). FL-1 (green) vs. FL-2 (red) dot plots were generated for the indicated groups. Cells with normal ΔΨm are detected as those with higher FL-2 signal (FL-1 bright , FL-2 bright cells)—indicative of higher levels of JC-1 aggregates (detected in FL-2, 570 nm). The loss of mitochondrial membrane potential results in increased levels of FL-1 bright , FL-2 dim cells—indicative of higher JC-1 monomer (detected in FL-1, 530 nm). g Apoptosis assay: the cells were subjected to Annexin V-FITC/Propidium Iodide (PI) double staining and data acquired using flow cytometry (BD FACS Calibur). FITC − /PI − cells indicate normal cells while FITC + /PI − , FITC + /PI + and PI + suggest the presence of cells in specific stages of apoptosis (early, late, or necrotic). The percentage of cells in individual quadrant is also indicated in the figure. h DNA content analysis: the cells were stained with propidium iodide and DNA content analysis (cell cycle) done employing flow cytometry (BD FACS Calibur). Histogram plot for specific groups is as shown. The relative fractions of cells in specific stages of cell cycle (G1, S or G2) are also indicated. H 2 O 2 treated group also showed distinctive sub-G1 population, indicating the presence of apoptotic cells.

    Techniques Used: Staining, Fluorescence, Whisker Assay, Flow Cytometry, Cytometry, Generated, Apoptosis Assay, Double Staining, FACS

    9) Product Images from "Activation of Piezo1 sensitizes cells to TRAIL-mediated apoptosis through mitochondrial outer membrane permeability"

    Article Title: Activation of Piezo1 sensitizes cells to TRAIL-mediated apoptosis through mitochondrial outer membrane permeability

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-019-2063-6

    Yoda1 and TRAIL induce mitochondrial dysfunction. a Representative flow plots of JC-1 assay after Yoda1 or DMSO and TRAIL treatment. b Percent of cells with depolarized mitochondria after DMSO or Yoda1 and TRAIL treatment ( n = 3). c Flow plots of MOMP due to DMSO or Yoda1 and TRAIL treatment. d Average MOMP of PC3 cells after treatment with DMSO or Yoda1 and TRAIL ( n = 3). e MOMP of PC3 cells treated with DMSO or Yoda1 and TRAIL at 1, 4, 8, 12, and 24 h timepoints ( n = 3). f Representative images of Bax activation of PC3 cells treated with DMSO or Yoda1 and TRAIL. The red channel is actin, green is active Bax, and blue is DAPI. Scale bars = 20 µm. g Fluorescent intensity of active Bax in PC3 cells treated with DMSO and TRAIL ( n = 57) or Yoda1 and TRAIL ( n = 40). h TRAIL sensitization of PC3 cells when treated with Yoda1 after scrambled siRNA, cytochrome c (CYCS) and Smac knockdown. a , c , f One representative experiment of three independent experiments. b , d , e , g , h Means and SD of three independent experiments. Statistical analysis was done using one-tailed ANOVA ( b , d ) and two-tailed unpaired t -test ( g , h ). * p
    Figure Legend Snippet: Yoda1 and TRAIL induce mitochondrial dysfunction. a Representative flow plots of JC-1 assay after Yoda1 or DMSO and TRAIL treatment. b Percent of cells with depolarized mitochondria after DMSO or Yoda1 and TRAIL treatment ( n = 3). c Flow plots of MOMP due to DMSO or Yoda1 and TRAIL treatment. d Average MOMP of PC3 cells after treatment with DMSO or Yoda1 and TRAIL ( n = 3). e MOMP of PC3 cells treated with DMSO or Yoda1 and TRAIL at 1, 4, 8, 12, and 24 h timepoints ( n = 3). f Representative images of Bax activation of PC3 cells treated with DMSO or Yoda1 and TRAIL. The red channel is actin, green is active Bax, and blue is DAPI. Scale bars = 20 µm. g Fluorescent intensity of active Bax in PC3 cells treated with DMSO and TRAIL ( n = 57) or Yoda1 and TRAIL ( n = 40). h TRAIL sensitization of PC3 cells when treated with Yoda1 after scrambled siRNA, cytochrome c (CYCS) and Smac knockdown. a , c , f One representative experiment of three independent experiments. b , d , e , g , h Means and SD of three independent experiments. Statistical analysis was done using one-tailed ANOVA ( b , d ) and two-tailed unpaired t -test ( g , h ). * p

    Techniques Used: Flow Cytometry, Activation Assay, One-tailed Test, Two Tailed Test

    10) Product Images from "A novel live-dead staining methodology to study malaria parasite viability"

    Article Title: A novel live-dead staining methodology to study malaria parasite viability

    Journal: Malaria Journal

    doi: 10.1186/1475-2875-12-190

    Living, dead and dying P. falciparum schizonts stained with JC-1. JC-1 staining of Plasmodium falciparum schizonts. The first row of images shows a living parasite as can be appreciated by the bright red signal at 568 nm indicating a functioning mitochondrion; the second row of images shows a dead parasite killed using anti-malarial drugs (no signal at 568 nm). In the third series only a faint red signal (sometimes difficult to detect) can be seen at 568 nm indicating that the mitochondrion membrane potential is disrupted and the parasite is dying.
    Figure Legend Snippet: Living, dead and dying P. falciparum schizonts stained with JC-1. JC-1 staining of Plasmodium falciparum schizonts. The first row of images shows a living parasite as can be appreciated by the bright red signal at 568 nm indicating a functioning mitochondrion; the second row of images shows a dead parasite killed using anti-malarial drugs (no signal at 568 nm). In the third series only a faint red signal (sometimes difficult to detect) can be seen at 568 nm indicating that the mitochondrion membrane potential is disrupted and the parasite is dying.

    Techniques Used: Staining

    Delayed death phenotypes monitored using JC-1 on synchronized P. falciparum FCR3 cultures exposed to chloroquine and the phospholipid pathway inhibitors prototype drug. P. falciparum FCR3 culture (5% hematocrit and 0.1 starting parasitaemia) were exposed to 7.82 nM chloroquine and the phospholipid pathway inhibitors prototype drugs (2.25 nM albitiazolium and 0.6 nM G25). After two hours of contact with the drugs, cell were washed and resuspended in drug-free fresh complete medium. JC-1 staining was then used to monitor parasite viability over the course of at least 3 cycles to detect any delayed drug effects. The results are expressed as means ± SEM (n = 3). Exposure of a ring culture to the IC 50 of chloroquine for 2 h has no delayed effect but acts on trophozoites and schizonts during the first cycle as expected (blue line), but exposure of the same ring culture to the IC 50 of G25 and albitiazolium for only 2 hours results in a potent delayed death effect, which for G25 reaches 100% within the second parasite cycle (red line) and for albitiazolium probably within beyond the third (green line) cycle.
    Figure Legend Snippet: Delayed death phenotypes monitored using JC-1 on synchronized P. falciparum FCR3 cultures exposed to chloroquine and the phospholipid pathway inhibitors prototype drug. P. falciparum FCR3 culture (5% hematocrit and 0.1 starting parasitaemia) were exposed to 7.82 nM chloroquine and the phospholipid pathway inhibitors prototype drugs (2.25 nM albitiazolium and 0.6 nM G25). After two hours of contact with the drugs, cell were washed and resuspended in drug-free fresh complete medium. JC-1 staining was then used to monitor parasite viability over the course of at least 3 cycles to detect any delayed drug effects. The results are expressed as means ± SEM (n = 3). Exposure of a ring culture to the IC 50 of chloroquine for 2 h has no delayed effect but acts on trophozoites and schizonts during the first cycle as expected (blue line), but exposure of the same ring culture to the IC 50 of G25 and albitiazolium for only 2 hours results in a potent delayed death effect, which for G25 reaches 100% within the second parasite cycle (red line) and for albitiazolium probably within beyond the third (green line) cycle.

    Techniques Used: Staining

    Living P. falciparum parasite stages stained with JC-1. Plasmodium falciparum blood stages (rings, trophozoites, schizonts and gametocytes) stained with JC-1. The first panel in each row shows the staining of the parasite’s nucleus with Hoechst dye (350 nm); the second panel shows JC-1 monomer visible at 488 nm (green channel), which colours the parasite’s cytoplasm; the third panel shows the JC-1 aggregate visible at 568 nm (red channel) staining the parasite’s active mitochondrion; while the last panel shows the corresponding bright field image.
    Figure Legend Snippet: Living P. falciparum parasite stages stained with JC-1. Plasmodium falciparum blood stages (rings, trophozoites, schizonts and gametocytes) stained with JC-1. The first panel in each row shows the staining of the parasite’s nucleus with Hoechst dye (350 nm); the second panel shows JC-1 monomer visible at 488 nm (green channel), which colours the parasite’s cytoplasm; the third panel shows the JC-1 aggregate visible at 568 nm (red channel) staining the parasite’s active mitochondrion; while the last panel shows the corresponding bright field image.

    Techniques Used: Staining

    Living Plamodium knowlesi and P. cynomolgi stages stained with JC-1. JC-1 staining of additional Plasmodium spp and stages. Top row: Plasmodium knowlesi in vitro cultured blood stage trophozoite. Middle row: Ex vivo harvested Plasmodium cynomolgi blood stage schizont. Bottom row: P. cynomolgi salivary gland sporozoite.
    Figure Legend Snippet: Living Plamodium knowlesi and P. cynomolgi stages stained with JC-1. JC-1 staining of additional Plasmodium spp and stages. Top row: Plasmodium knowlesi in vitro cultured blood stage trophozoite. Middle row: Ex vivo harvested Plasmodium cynomolgi blood stage schizont. Bottom row: P. cynomolgi salivary gland sporozoite.

    Techniques Used: Staining, In Vitro, Cell Culture, Ex Vivo

    IC 50 determination using JC-1 and/or pLDH in parallel on drug treated P. falciparum cultures. Culture P. falciparum -infected erythrocyte were incubated for 48 h in the presence of the indicated drug concentration at haematocrit of 5% and 1% parasitaemia. Panel A - shows that the inter-experiment variability between 2 separate JC-1 determinations is low (red and violet lines) and that the methodology is more accurate (IC 50 is consistently lower) than the IC 50 determination obtained with the pLDH methodology; Panel B - shows typical curves obtained by splitting parasites derived from a same mother culture into plates, in which they were cultured in parallel under the same environmental conditions in the presence of rising artemisinin concentrations and IC 50 curves where either determined using the pLDH (violet line) or the JC-1 (blue line) methodology; Panel C – shows typical curves obtained by splitting parasites derived from a same mother culture into plates, in which they were cultured in parallel under the same environmental conditions in the presence of rising atovaquone concentrations and IC 50 curves where either determined using the pLDH (violet line) or the JC-1 (blue line) methodology.
    Figure Legend Snippet: IC 50 determination using JC-1 and/or pLDH in parallel on drug treated P. falciparum cultures. Culture P. falciparum -infected erythrocyte were incubated for 48 h in the presence of the indicated drug concentration at haematocrit of 5% and 1% parasitaemia. Panel A - shows that the inter-experiment variability between 2 separate JC-1 determinations is low (red and violet lines) and that the methodology is more accurate (IC 50 is consistently lower) than the IC 50 determination obtained with the pLDH methodology; Panel B - shows typical curves obtained by splitting parasites derived from a same mother culture into plates, in which they were cultured in parallel under the same environmental conditions in the presence of rising artemisinin concentrations and IC 50 curves where either determined using the pLDH (violet line) or the JC-1 (blue line) methodology; Panel C – shows typical curves obtained by splitting parasites derived from a same mother culture into plates, in which they were cultured in parallel under the same environmental conditions in the presence of rising atovaquone concentrations and IC 50 curves where either determined using the pLDH (violet line) or the JC-1 (blue line) methodology.

    Techniques Used: Infection, Incubation, Concentration Assay, Derivative Assay, Cell Culture

    11) Product Images from "Cabbage (Brassica oleracea var. capitata) Protects against H2O2-Induced Oxidative Stress by Preventing Mitochondrial Dysfunction in H9c2 Cardiomyoblasts"

    Article Title: Cabbage (Brassica oleracea var. capitata) Protects against H2O2-Induced Oxidative Stress by Preventing Mitochondrial Dysfunction in H9c2 Cardiomyoblasts

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2018/2179021

    Cabbage extract prevents mitochondrial dysfunction upon H 2 O 2 -induced oxidative stress in H9c2 cardiomyocytes. The MMP was determined by incubating cells with JC-1 for 20 min. (a) Representative images of and (b) fluorescence intensities in H9c2 cardiomyocytes treated with 500 μ M H 2 O 2 for 24 h following pretreatment with 100, 200, and 300 μ g/ml cabbage extract for 24 h. (c) Quantitative RT-PCR analysis of mitochondrial biogenesis-related genes (NRF-1, PPAR α , ERR α , and PGC-1 β ). Western blot analysis was performed in triplicate with three independent samples. Data are expressed as fold changes ± SEM versus control cells. Significance was analyzed by a one-way ANOVA followed by the Bonferroni post hoc test. ## P
    Figure Legend Snippet: Cabbage extract prevents mitochondrial dysfunction upon H 2 O 2 -induced oxidative stress in H9c2 cardiomyocytes. The MMP was determined by incubating cells with JC-1 for 20 min. (a) Representative images of and (b) fluorescence intensities in H9c2 cardiomyocytes treated with 500 μ M H 2 O 2 for 24 h following pretreatment with 100, 200, and 300 μ g/ml cabbage extract for 24 h. (c) Quantitative RT-PCR analysis of mitochondrial biogenesis-related genes (NRF-1, PPAR α , ERR α , and PGC-1 β ). Western blot analysis was performed in triplicate with three independent samples. Data are expressed as fold changes ± SEM versus control cells. Significance was analyzed by a one-way ANOVA followed by the Bonferroni post hoc test. ## P

    Techniques Used: Fluorescence, Quantitative RT-PCR, Pyrolysis Gas Chromatography, Western Blot

    12) Product Images from "Suppression of human arthritis synovial fibroblasts inflammation using dexamethasone-carbon nanotubes via increasing caveolin-dependent endocytosis and recovering mitochondrial membrane potential"

    Article Title: Suppression of human arthritis synovial fibroblasts inflammation using dexamethasone-carbon nanotubes via increasing caveolin-dependent endocytosis and recovering mitochondrial membrane potential

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S142122

    Effect of DEX-PEG-coated CNT on mitochondrial membrane disruption in TNF-α-stimulated FLS. Notes: ( A , B ) JC-1 staining showed depolarized mitochondria (green, J-monomer) and polarized mitochondria (red, J-aggregate) membrane potentials after 24 h. H 2 O 2 (50 μM) was used as a positive control. Scale bar: 20 μm. PEG-coated CNT (1 μg/mL) showed no significant changes in the greater level of mitochondrial membrane disruption (green). DEX-PEG-coated CNT inhibited mitochondrial membrane disruption at low doses (0.25 μg/mL) compared to DEX (1 μg/mL) in TNF-α-stimulated FLS. All data represent mean ± SEM (n=6). *** P
    Figure Legend Snippet: Effect of DEX-PEG-coated CNT on mitochondrial membrane disruption in TNF-α-stimulated FLS. Notes: ( A , B ) JC-1 staining showed depolarized mitochondria (green, J-monomer) and polarized mitochondria (red, J-aggregate) membrane potentials after 24 h. H 2 O 2 (50 μM) was used as a positive control. Scale bar: 20 μm. PEG-coated CNT (1 μg/mL) showed no significant changes in the greater level of mitochondrial membrane disruption (green). DEX-PEG-coated CNT inhibited mitochondrial membrane disruption at low doses (0.25 μg/mL) compared to DEX (1 μg/mL) in TNF-α-stimulated FLS. All data represent mean ± SEM (n=6). *** P

    Techniques Used: Staining, Positive Control

    13) Product Images from "A Rapid and Convenient Method for in Vivo Fluorescent Imaging of Protoscolices of Echinococcus multilocularis"

    Article Title: A Rapid and Convenient Method for in Vivo Fluorescent Imaging of Protoscolices of Echinococcus multilocularis

    Journal: The Korean Journal of Parasitology

    doi: 10.3347/kjp.2016.54.2.225

    Effect of Met and its combination with ABZSO on viability and mitochondrial function of protoscolices of E. multilocularis . (A) Viability of protoscolices incubated for 2 days, respective with varieties of Met and ABZSO concentration, in histograms. (B) Representative confocal images showing JC-1 fluorescence in protoscolices incubated under control conditions or treated with 10 mM Met or 10 mM Met combined with 15 µM ABZSO for 12 hr (scale bar indicates 100 μm). (C) Histograms showing the values of the red/green JC-1 fluorescence ratios measured in 3 groups of parasites by Image J Software. (D) Red and green fluorescence of 3 groups of protoscolices. The changing trend of fluorescence signal of control protoscolices (Con; ●), Met-treated protoscolices (Met; ■), and Met combined with ABZSO-treated protoscolices (Met+ABZSO; ▲) as arbitrary units (AU) with the change of time. Experiments were carried out with 2×10 3 parasites by triplicate, and error bars represent SDs. Comparison of fluorescent intensity between any 2 groups of parasites at different time points was made. * P
    Figure Legend Snippet: Effect of Met and its combination with ABZSO on viability and mitochondrial function of protoscolices of E. multilocularis . (A) Viability of protoscolices incubated for 2 days, respective with varieties of Met and ABZSO concentration, in histograms. (B) Representative confocal images showing JC-1 fluorescence in protoscolices incubated under control conditions or treated with 10 mM Met or 10 mM Met combined with 15 µM ABZSO for 12 hr (scale bar indicates 100 μm). (C) Histograms showing the values of the red/green JC-1 fluorescence ratios measured in 3 groups of parasites by Image J Software. (D) Red and green fluorescence of 3 groups of protoscolices. The changing trend of fluorescence signal of control protoscolices (Con; ●), Met-treated protoscolices (Met; ■), and Met combined with ABZSO-treated protoscolices (Met+ABZSO; ▲) as arbitrary units (AU) with the change of time. Experiments were carried out with 2×10 3 parasites by triplicate, and error bars represent SDs. Comparison of fluorescent intensity between any 2 groups of parasites at different time points was made. * P

    Techniques Used: Incubation, Concentration Assay, Fluorescence, Software

    Fluorescent imaging was performed on BALB/c mice (n=10) challenged with 3×10 3 control protoscolices, Met-treated protoscolices or Met combined with ABZSO-treated protoscolices after 1 min of inoculation. JC-1 fluorescence was measured using an in vivo small-animal imaging system. (A) Representative images of individual mouse from each group. Quantified data represented radiant efficiency for parasites in p/sec/cm 2 /sr/μW/cm 2 . (B, C) Histograms showing the values of the red/green JC-1 fluorescence ratios measured in control, Met-treated protoscolices and Met combined with ABZSO treated protoscolices. ** P
    Figure Legend Snippet: Fluorescent imaging was performed on BALB/c mice (n=10) challenged with 3×10 3 control protoscolices, Met-treated protoscolices or Met combined with ABZSO-treated protoscolices after 1 min of inoculation. JC-1 fluorescence was measured using an in vivo small-animal imaging system. (A) Representative images of individual mouse from each group. Quantified data represented radiant efficiency for parasites in p/sec/cm 2 /sr/μW/cm 2 . (B, C) Histograms showing the values of the red/green JC-1 fluorescence ratios measured in control, Met-treated protoscolices and Met combined with ABZSO treated protoscolices. ** P

    Techniques Used: Imaging, Mouse Assay, Fluorescence, In Vivo, Size-exclusion Chromatography

    14) Product Images from "Immunotoxicity of titanium dioxide nanoparticles via simultaneous induction of apoptosis and multiple toll-like receptors signaling through ROS-dependent SAPK/JNK and p38 MAPK activation"

    Article Title: Immunotoxicity of titanium dioxide nanoparticles via simultaneous induction of apoptosis and multiple toll-like receptors signaling through ROS-dependent SAPK/JNK and p38 MAPK activation

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S176087

    TiO 2 NPs induced the disruption of MMP (Δψ m ) of RAW 264.7 cells. Notes: Cells were incubated with indicated concentrations of TiO 2 NPs for 24 hours and incubated with 5 µM of JC-1 for 15 minutes, loaded on NC-Slide A8 and analyzed using fluorescence microscopy NucleoCounter ® NC-3000™. Cellular JC-1 monomers and aggregates are detected as green and red fluorescence, respectively. Untreated cells were considered as control in the experiment. Magnification 200×. Abbreviations: TiO 2 , titanium dioxide; MMP, mitochondrial membrane potential; NPs, nanoparticles.
    Figure Legend Snippet: TiO 2 NPs induced the disruption of MMP (Δψ m ) of RAW 264.7 cells. Notes: Cells were incubated with indicated concentrations of TiO 2 NPs for 24 hours and incubated with 5 µM of JC-1 for 15 minutes, loaded on NC-Slide A8 and analyzed using fluorescence microscopy NucleoCounter ® NC-3000™. Cellular JC-1 monomers and aggregates are detected as green and red fluorescence, respectively. Untreated cells were considered as control in the experiment. Magnification 200×. Abbreviations: TiO 2 , titanium dioxide; MMP, mitochondrial membrane potential; NPs, nanoparticles.

    Techniques Used: Incubation, Fluorescence, Microscopy

    15) Product Images from "Probing Novel Roles of the Mitochondrial Uniporter in Ovarian Cancer Cells Using Nanoparticles *Probing Novel Roles of the Mitochondrial Uniporter in Ovarian Cancer Cells Using Nanoparticles * ♦"

    Article Title: Probing Novel Roles of the Mitochondrial Uniporter in Ovarian Cancer Cells Using Nanoparticles *Probing Novel Roles of the Mitochondrial Uniporter in Ovarian Cancer Cells Using Nanoparticles * ♦

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.435206

    MICU1 modulates ΔΨ m responses in malignant cells. A and B , in A2780 cells loaded with the fluorescent ΔΨ m indicator DiBASC 2 (3), + AuNPs caused rapid and substantial membrane depolarization ( A ), whereas nonmalignant OSE cells showed slight hyperpolarization ( B ). + AuNP effects on ΔΨ m in A2780 cells were enhanced by MICU1 blockade (siRNA). NT , nontreated controls. C–F , these + AuNP effects on ΔΨ m were also evident in confocal images of the fluorescent dye JC-1 in malignant cells ( C ) versus normal cells ( D ) and corresponding measurements of JC-1 ( E and F ). Inhibition of uniporter by Ru360 enhanced + AuNP effects on ΔΨ m . A similar effect was observed with MICU1 siRNA (not shown). Scale bar = 20 μm; n = 3. w/o + AuNP , without + AuNP.
    Figure Legend Snippet: MICU1 modulates ΔΨ m responses in malignant cells. A and B , in A2780 cells loaded with the fluorescent ΔΨ m indicator DiBASC 2 (3), + AuNPs caused rapid and substantial membrane depolarization ( A ), whereas nonmalignant OSE cells showed slight hyperpolarization ( B ). + AuNP effects on ΔΨ m in A2780 cells were enhanced by MICU1 blockade (siRNA). NT , nontreated controls. C–F , these + AuNP effects on ΔΨ m were also evident in confocal images of the fluorescent dye JC-1 in malignant cells ( C ) versus normal cells ( D ) and corresponding measurements of JC-1 ( E and F ). Inhibition of uniporter by Ru360 enhanced + AuNP effects on ΔΨ m . A similar effect was observed with MICU1 siRNA (not shown). Scale bar = 20 μm; n = 3. w/o + AuNP , without + AuNP.

    Techniques Used: Inhibition

    16) Product Images from "Intrinsic mitochondrial DNA repair defects in Ataxia Telangiectasia"

    Article Title: Intrinsic mitochondrial DNA repair defects in Ataxia Telangiectasia

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2013.11.002

    Basal levels of mtDNA damage are increased in the absence of ATM leading to mitochondrial dysfunction. Genomic DNA was isolated from ATM-proficient and deficient samples and mtDNA integrity was analyzed using QPCR. Data were normalized to mtDNA content using amplification of the small mtDNA fragment (Santos et al., 2006). DNA was isolated 3 independent times and QPCR was performed two independent times with each DNA isolate, error bars represent ± SEM; (A) patient-derived cells and (B) mouse tissue (C) siRNA was transiently transfected in WT cells and levels of ATM analyzed by immunoblots 4 days after transfections (left). MtDNA damage was estimated in cells transfected with scrambled-control or siRNA targeted to ATM (right). QPCR was performed two independent times for each DNA sample from 3 independent experiments, error bars represent ± SEM. WT and A-T cells were submitted to 60 min treatment with 200 μM H 2 O 2 . Immediately after treatment cells were collected and: (D) mitochondrial ROS measured using Mitosox by FACS, N=3; * indicates significance between control WT and control A-T, and between A-T control and treated samples; (E) mitochondrial membrane polarization judged using JC-1 by FACS, N=3, * indicates significance using WT control as reference or between A-T control and treated cells. (F) Steady state levels of ATP were estimated using a luciferase-based assay. Experiments were reproduced at least 3 independent times; error bars represent ± SD. = p ≤0.05. * indicates significance between WT and A-T control. (G) Doubling time was calculated by following cell growth and counting cells each time they reached confluency. Results represent the mean doubling time calculated over a period of 3 weeks for two independently grown cultures for each cell type.
    Figure Legend Snippet: Basal levels of mtDNA damage are increased in the absence of ATM leading to mitochondrial dysfunction. Genomic DNA was isolated from ATM-proficient and deficient samples and mtDNA integrity was analyzed using QPCR. Data were normalized to mtDNA content using amplification of the small mtDNA fragment (Santos et al., 2006). DNA was isolated 3 independent times and QPCR was performed two independent times with each DNA isolate, error bars represent ± SEM; (A) patient-derived cells and (B) mouse tissue (C) siRNA was transiently transfected in WT cells and levels of ATM analyzed by immunoblots 4 days after transfections (left). MtDNA damage was estimated in cells transfected with scrambled-control or siRNA targeted to ATM (right). QPCR was performed two independent times for each DNA sample from 3 independent experiments, error bars represent ± SEM. WT and A-T cells were submitted to 60 min treatment with 200 μM H 2 O 2 . Immediately after treatment cells were collected and: (D) mitochondrial ROS measured using Mitosox by FACS, N=3; * indicates significance between control WT and control A-T, and between A-T control and treated samples; (E) mitochondrial membrane polarization judged using JC-1 by FACS, N=3, * indicates significance using WT control as reference or between A-T control and treated cells. (F) Steady state levels of ATP were estimated using a luciferase-based assay. Experiments were reproduced at least 3 independent times; error bars represent ± SD. = p ≤0.05. * indicates significance between WT and A-T control. (G) Doubling time was calculated by following cell growth and counting cells each time they reached confluency. Results represent the mean doubling time calculated over a period of 3 weeks for two independently grown cultures for each cell type.

    Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Amplification, Derivative Assay, Transfection, Western Blot, FACS, Luciferase

    17) Product Images from "NOX4 modulates macrophage phenotype and mitochondrial biogenesis in asbestosis"

    Article Title: NOX4 modulates macrophage phenotype and mitochondrial biogenesis in asbestosis

    Journal: JCI Insight

    doi: 10.1172/jci.insight.126551

    NOX4 regulates mitochondrial ROS and bioenergetics. ( A and B ) H 2 O 2 generation in isolated mitochondria of lung macrophages from normal ( n = 5) or asbestosis subjects ( n = 6). ( C ) ROS production of lung macrophages from Nox4 fl/fl and Nox4 –/– Lyz2-Cre mice ( n = 3) exposed to either MMVF or chrysotile asbestos measured by MitoSOX. ( D ) Mitochondrial membrane potential measured in BMDMs from WT and Nox4 –/– mice by JC-1. Red, JC-1 aggregates; green, JC-1 monomer; overlay, JC-1 red and green fluorescent overlay. ( E ) Quantification of JC-1 green/red fluorescence ratio ( n = 3). ( F ) Oxygen consumption rate (OCR) tracing, ( G ) basal OCR, and ( H ) maximal OCR of lung macrophages from WT and Nox4 –/– mice exposed to either MMVF or chrysotile asbestos ( n = 5). * P
    Figure Legend Snippet: NOX4 regulates mitochondrial ROS and bioenergetics. ( A and B ) H 2 O 2 generation in isolated mitochondria of lung macrophages from normal ( n = 5) or asbestosis subjects ( n = 6). ( C ) ROS production of lung macrophages from Nox4 fl/fl and Nox4 –/– Lyz2-Cre mice ( n = 3) exposed to either MMVF or chrysotile asbestos measured by MitoSOX. ( D ) Mitochondrial membrane potential measured in BMDMs from WT and Nox4 –/– mice by JC-1. Red, JC-1 aggregates; green, JC-1 monomer; overlay, JC-1 red and green fluorescent overlay. ( E ) Quantification of JC-1 green/red fluorescence ratio ( n = 3). ( F ) Oxygen consumption rate (OCR) tracing, ( G ) basal OCR, and ( H ) maximal OCR of lung macrophages from WT and Nox4 –/– mice exposed to either MMVF or chrysotile asbestos ( n = 5). * P

    Techniques Used: Isolation, Mouse Assay, Fluorescence

    18) Product Images from "Late life metformin treatment limits cell survival and shortens lifespan by triggering an aging-associated failure of energy metabolism"

    Article Title: Late life metformin treatment limits cell survival and shortens lifespan by triggering an aging-associated failure of energy metabolism

    Journal: bioRxiv

    doi: 10.1101/863357

    Ectopic ATP supplementation alleviates the toxicity of ETC complex I inhibitor rotenone in human primary fibroblasts. Pre-senescent primary human skin fibroblasts were treated with indicated doses of rotenone for 21 hours; cell survival ( A , MTT assay), cell death ( B , LDH assay), and mitochondrial membrane potential ( C , JC-1 assay) were measured. Values relative to the respective untreated control (no ATP, no rotenone) are shown for all assays. n=3, mean and SEM are presented, two-tailed unpaired t-test was used for the statistical analysis, statistical values are shown in Table S2 ; * p
    Figure Legend Snippet: Ectopic ATP supplementation alleviates the toxicity of ETC complex I inhibitor rotenone in human primary fibroblasts. Pre-senescent primary human skin fibroblasts were treated with indicated doses of rotenone for 21 hours; cell survival ( A , MTT assay), cell death ( B , LDH assay), and mitochondrial membrane potential ( C , JC-1 assay) were measured. Values relative to the respective untreated control (no ATP, no rotenone) are shown for all assays. n=3, mean and SEM are presented, two-tailed unpaired t-test was used for the statistical analysis, statistical values are shown in Table S2 ; * p

    Techniques Used: MTT Assay, Lactate Dehydrogenase Assay, Two Tailed Test

    Rapamycin co-treatment alleviates metformin toxicity in human primary cells. Pre-senescent (PD44) primary human skin fibroblasts were treated with indicated doses of metformin in presence or absence of indicated concentrations of rapamycin (Rap) for 24 ( A, B ) and 20 ( C, D ) hours. Cell death ( A , LDH assay), cell survival ( B , MTT assay), ATP content ( C ) and mitochondrial membrane potential ( D , JC-1 staining) were measured; the data are complementary to Fig. 7A-C ; values are relative to the respective untreated control (no rapamycin, no metformin) for each assay. n=3, mean and SEM are presented, two-tailed unpaired t-test was used for the statistical analysis, statistical values are shown in Table S2 ; * p
    Figure Legend Snippet: Rapamycin co-treatment alleviates metformin toxicity in human primary cells. Pre-senescent (PD44) primary human skin fibroblasts were treated with indicated doses of metformin in presence or absence of indicated concentrations of rapamycin (Rap) for 24 ( A, B ) and 20 ( C, D ) hours. Cell death ( A , LDH assay), cell survival ( B , MTT assay), ATP content ( C ) and mitochondrial membrane potential ( D , JC-1 staining) were measured; the data are complementary to Fig. 7A-C ; values are relative to the respective untreated control (no rapamycin, no metformin) for each assay. n=3, mean and SEM are presented, two-tailed unpaired t-test was used for the statistical analysis, statistical values are shown in Table S2 ; * p

    Techniques Used: Lactate Dehydrogenase Assay, MTT Assay, Staining, Two Tailed Test

    Metformin toxicity associates with loss of mitochondrial homeostasis and ATP exhaustion in human cells and nematodes. Young (PD36) and old (PD61) human skin fibroblasts were treated with metformin for 15 ( F, G ) or 20 ( A, B ) hours; cell survival ( A, MTT assay), cell death ( B, LDH assay), mitochondrial membrane potential ( G, JC-1 assay) and ATP content ( F ) were measured; all values are relative to the untreated control of a given age. ( C – E ) Young (PD29) and old (PD61) primary human skin fibroblasts were treated with metformin for 16h, then washed and pre-incubated with media containing high glucose (10mM), glutamine (2mM) and pyruvate (1mM); continuous oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were recorded following injection of oligomycin (1μM), FCCP (3μM), antimycin-A (0,5μM) and rotenone (0,5μM) (shown in Fig S4 ). The recorded values were normalized to protein content, basal respiration ( C ), maximal respiration ( D ) and basal ECAR ( E ) were quantified. ( H ) Young (adulthood day 1) and old (adulthood day 10) wild type nematodes were treated with 50mM metformin, whole organism ATP levels were measured after 36h. Pre-senescent (PD44) fibroblasts were treated with metformin for 24h in presence or absence of ATP; ATP content ( I ) and cell survival ( J ) were measured; all values are relative to the untreated control (no metformin, no ATP) for each assay. For H n=100, for all other panels n=3, mean and SEM are presented, two-tailed unpaired t-test was used for the statistical analysis, all statistical values are presented in Table S2 ; * p
    Figure Legend Snippet: Metformin toxicity associates with loss of mitochondrial homeostasis and ATP exhaustion in human cells and nematodes. Young (PD36) and old (PD61) human skin fibroblasts were treated with metformin for 15 ( F, G ) or 20 ( A, B ) hours; cell survival ( A, MTT assay), cell death ( B, LDH assay), mitochondrial membrane potential ( G, JC-1 assay) and ATP content ( F ) were measured; all values are relative to the untreated control of a given age. ( C – E ) Young (PD29) and old (PD61) primary human skin fibroblasts were treated with metformin for 16h, then washed and pre-incubated with media containing high glucose (10mM), glutamine (2mM) and pyruvate (1mM); continuous oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were recorded following injection of oligomycin (1μM), FCCP (3μM), antimycin-A (0,5μM) and rotenone (0,5μM) (shown in Fig S4 ). The recorded values were normalized to protein content, basal respiration ( C ), maximal respiration ( D ) and basal ECAR ( E ) were quantified. ( H ) Young (adulthood day 1) and old (adulthood day 10) wild type nematodes were treated with 50mM metformin, whole organism ATP levels were measured after 36h. Pre-senescent (PD44) fibroblasts were treated with metformin for 24h in presence or absence of ATP; ATP content ( I ) and cell survival ( J ) were measured; all values are relative to the untreated control (no metformin, no ATP) for each assay. For H n=100, for all other panels n=3, mean and SEM are presented, two-tailed unpaired t-test was used for the statistical analysis, all statistical values are presented in Table S2 ; * p

    Techniques Used: MTT Assay, Lactate Dehydrogenase Assay, Incubation, Injection, Two Tailed Test

    Mitochondrial impairments pre-dispose nematodes and human cells to metformin toxicity. ( A ) WT animals ( left panel ) and ubl-5(ok3389) mutants ( right panel ) were treated with 50mM metformin on days 1 and 10 of adulthood (AD1 and AD10 respectively), survival was scored daily. ( B ) Early passage (population doubling, PD37) fibroblasts were treated with indicated doses of metformin (Met) in absence or presence of indicated concentrations of FCCP for 24 hours; mitochondrial membrane potential was measured by JC-1 staining. All values are relative to the untreated control (no FCCP, no metformin). DMSO was used as vehicle control for FCCP, the own effect of the vehicle is seen at 0μM FCCP. Grey bars depict the effect of FCCP alone (metformin-independent) on the mitochondrial membrane potential (MMP), proof of concept MMP decline is seen at 5μM FCCP. For A significance was calculated by log-rank test, all n numbers (n≥100 in all cases) and statistical values are presented in Table S1 ; for B n=3, mean and SEM are presented; two-tailed unpaired t-test was used for the statistical analysis, statistical values are shown in Table S2 ; **** p
    Figure Legend Snippet: Mitochondrial impairments pre-dispose nematodes and human cells to metformin toxicity. ( A ) WT animals ( left panel ) and ubl-5(ok3389) mutants ( right panel ) were treated with 50mM metformin on days 1 and 10 of adulthood (AD1 and AD10 respectively), survival was scored daily. ( B ) Early passage (population doubling, PD37) fibroblasts were treated with indicated doses of metformin (Met) in absence or presence of indicated concentrations of FCCP for 24 hours; mitochondrial membrane potential was measured by JC-1 staining. All values are relative to the untreated control (no FCCP, no metformin). DMSO was used as vehicle control for FCCP, the own effect of the vehicle is seen at 0μM FCCP. Grey bars depict the effect of FCCP alone (metformin-independent) on the mitochondrial membrane potential (MMP), proof of concept MMP decline is seen at 5μM FCCP. For A significance was calculated by log-rank test, all n numbers (n≥100 in all cases) and statistical values are presented in Table S1 ; for B n=3, mean and SEM are presented; two-tailed unpaired t-test was used for the statistical analysis, statistical values are shown in Table S2 ; **** p

    Techniques Used: Staining, Two Tailed Test

    Ectopic ATP supplementation alleviates metformin toxicity in human primary fibroblasts. Pre-senescent (PD44) primary human skin fibroblasts were treated with indicated doses of metformin in presence or absence of indicated concentrations of ATP for 24h hours; cell death ( A , LDH assay), cell survival ( B , MTT assay), ATP content ( C ) and mitochondrial membrane potential ( D , JC-1 assay) were measured; the data are complementary to Fig. 3I-J ; values are relative to the respective untreated control (no ATP, no metformin) for each assay. The data from cells treated with 5mM metformin demonstrates that ATP exhaustion and loss of mitochondrial membrane potential precede metformin-triggered cell death. n=3, mean and SEM are presented, two-tailed unpaired t-test was used for the statistical analysis, statistical values are shown in Table S2 ; * p
    Figure Legend Snippet: Ectopic ATP supplementation alleviates metformin toxicity in human primary fibroblasts. Pre-senescent (PD44) primary human skin fibroblasts were treated with indicated doses of metformin in presence or absence of indicated concentrations of ATP for 24h hours; cell death ( A , LDH assay), cell survival ( B , MTT assay), ATP content ( C ) and mitochondrial membrane potential ( D , JC-1 assay) were measured; the data are complementary to Fig. 3I-J ; values are relative to the respective untreated control (no ATP, no metformin) for each assay. The data from cells treated with 5mM metformin demonstrates that ATP exhaustion and loss of mitochondrial membrane potential precede metformin-triggered cell death. n=3, mean and SEM are presented, two-tailed unpaired t-test was used for the statistical analysis, statistical values are shown in Table S2 ; * p

    Techniques Used: Lactate Dehydrogenase Assay, MTT Assay, Two Tailed Test

    19) Product Images from "Decreased Uptake and Enhanced Mitochondrial Protection Underlie Reduced Toxicity of Nanoceria in Human Monocyte-Derived Macrophages"

    Article Title: Decreased Uptake and Enhanced Mitochondrial Protection Underlie Reduced Toxicity of Nanoceria in Human Monocyte-Derived Macrophages

    Journal: Journal of biomedical nanotechnology

    doi: 10.1166/jbn.2016.2320

    Nanoceria does not induce cytotoxicity and mitochondrial damage in human MDMs A) MDMs were exposed to 0–20 µg/mL nanoceria for 24 or 48 hours and cell death was quantified by flow cytometry (propidium iodide staining). B) MDMs were exposed to 20 µg/mL nanoceria for 24 or 48 hours, labeled with JC-1 probe, and analyzed by fluorescent microscopy. C) MDMs were immunostained with an antibody against activated Bax (6A7) and analyzed by fluorescent microscopy. D) MDMs were labeled for AIF protein and analyzed by flow cytometry. H 2 O 2 and Staurosporine (STS) were used as positive controls. Data are represented as mean ± SEM of at least 3–6 independent experiments with triplicates of each condition. Data were analyzed by analysis of variance (ANOVA) followed by Tukey’s post hoc test or students t-test. **p
    Figure Legend Snippet: Nanoceria does not induce cytotoxicity and mitochondrial damage in human MDMs A) MDMs were exposed to 0–20 µg/mL nanoceria for 24 or 48 hours and cell death was quantified by flow cytometry (propidium iodide staining). B) MDMs were exposed to 20 µg/mL nanoceria for 24 or 48 hours, labeled with JC-1 probe, and analyzed by fluorescent microscopy. C) MDMs were immunostained with an antibody against activated Bax (6A7) and analyzed by fluorescent microscopy. D) MDMs were labeled for AIF protein and analyzed by flow cytometry. H 2 O 2 and Staurosporine (STS) were used as positive controls. Data are represented as mean ± SEM of at least 3–6 independent experiments with triplicates of each condition. Data were analyzed by analysis of variance (ANOVA) followed by Tukey’s post hoc test or students t-test. **p

    Techniques Used: Flow Cytometry, Cytometry, Staining, Labeling, Microscopy

    20) Product Images from "Neuroprotective effects of seaweeds against 6-hydroxidopamine-induced cell death on an in vitro human neuroblastoma model"

    Article Title: Neuroprotective effects of seaweeds against 6-hydroxidopamine-induced cell death on an in vitro human neuroblastoma model

    Journal: BMC Complementary and Alternative Medicine

    doi: 10.1186/s12906-018-2103-2

    6-OHDA (100 μM) effects in the presence or absence of seaweeds extracts (1 mg/mL) in mitochondrial membrane potential of SH-SY5Y cells after 3 h ( a ) and 6 h ( b ) of incubation. The results were obtained by the ratio between the monomers/aggregates of JC-1. The values in each column represent the mean ± standard error of the mean (SEM) of 3 or 4 independent experiments. Symbols represent statistically significant differences ( p
    Figure Legend Snippet: 6-OHDA (100 μM) effects in the presence or absence of seaweeds extracts (1 mg/mL) in mitochondrial membrane potential of SH-SY5Y cells after 3 h ( a ) and 6 h ( b ) of incubation. The results were obtained by the ratio between the monomers/aggregates of JC-1. The values in each column represent the mean ± standard error of the mean (SEM) of 3 or 4 independent experiments. Symbols represent statistically significant differences ( p

    Techniques Used: Incubation

    21) Product Images from "ATF6α regulates morphological changes associated with senescence in human fibroblasts"

    Article Title: ATF6α regulates morphological changes associated with senescence in human fibroblasts

    Journal: Oncotarget

    doi: 10.18632/oncotarget.11505

    Control of some markers of senescence by the UPR A. Exponentially growing or senescent NHDFs were subjected to PERK, ATF6α or IRE1α silencing by siRNA as well as a non-target control. Cells were counted 4 days after the siRNA transfection and the population doublings were calculated. The bar chart represents the mean ± SD of the count of three independent culture dishes. B. The percentage of SA-β-Gal-positive cells 4 days after the siRNA transfection is indicated. SA-β-Gal positive cells were counted in 3 independent microscopic fields. The bar chart represents the mean ± SD of each 3 counts. This experiment is representative of 2 independent ones C. NHDFs were treated as in (A), counted 4 days after the siRNA transfection, and subjected to flow cytometry analysis to assess their granularity according to their side scatter factor (SSC-A). Bar chart represents the percentage of cells in the subpopulation with the highest granularity. The histograms represent the mean ± SD of the 3 counts. This experiment is representative of 3 independent ones. D. Representative confocal microscopic images of JC1 staining performed in exponentially growing and senescent NHDFs. Bar represents 50 μm. E. NHDFs were treated as in (A) at different population doubling as indicated, stained with JC-1. A Bar chart representing the JC-1 red/green ratio is given. This experiment represents the mean ± SD of 3 independent measures. F. Senescent NHDFs were treated as in A and the senescent markers DCR2, IL6, CCL2 were measured at the mRNA levels by qRT-PCR and were normalized to Rplp0 levels. Results were performed in triplicate. The bar chart represents the mean ± SD of the 3 measures.
    Figure Legend Snippet: Control of some markers of senescence by the UPR A. Exponentially growing or senescent NHDFs were subjected to PERK, ATF6α or IRE1α silencing by siRNA as well as a non-target control. Cells were counted 4 days after the siRNA transfection and the population doublings were calculated. The bar chart represents the mean ± SD of the count of three independent culture dishes. B. The percentage of SA-β-Gal-positive cells 4 days after the siRNA transfection is indicated. SA-β-Gal positive cells were counted in 3 independent microscopic fields. The bar chart represents the mean ± SD of each 3 counts. This experiment is representative of 2 independent ones C. NHDFs were treated as in (A), counted 4 days after the siRNA transfection, and subjected to flow cytometry analysis to assess their granularity according to their side scatter factor (SSC-A). Bar chart represents the percentage of cells in the subpopulation with the highest granularity. The histograms represent the mean ± SD of the 3 counts. This experiment is representative of 3 independent ones. D. Representative confocal microscopic images of JC1 staining performed in exponentially growing and senescent NHDFs. Bar represents 50 μm. E. NHDFs were treated as in (A) at different population doubling as indicated, stained with JC-1. A Bar chart representing the JC-1 red/green ratio is given. This experiment represents the mean ± SD of 3 independent measures. F. Senescent NHDFs were treated as in A and the senescent markers DCR2, IL6, CCL2 were measured at the mRNA levels by qRT-PCR and were normalized to Rplp0 levels. Results were performed in triplicate. The bar chart represents the mean ± SD of the 3 measures.

    Techniques Used: Transfection, Flow Cytometry, Cytometry, Staining, Quantitative RT-PCR

    22) Product Images from "The pro-inflammatory phenotype of the human non-classical monocyte subset is attributed to senescence"

    Article Title: The pro-inflammatory phenotype of the human non-classical monocyte subset is attributed to senescence

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0327-1

    Non-classical monocytes exhibit several features of senescence. a Relative total cellular ROS levels as measured using H 2 DCFDA ROS indicator. b Relative mitochondrial ROS levels as measured using MitoSOX mitochondrial superoxide indicator. c – d Relative mitochondrial membrane potential (MMP) as measured using DIOC 6 and JC-1. e Relative expression levels of p-ERK. All the parameters were measured using flow cytometry. Each line represents one donor; n ≥ 3. * p
    Figure Legend Snippet: Non-classical monocytes exhibit several features of senescence. a Relative total cellular ROS levels as measured using H 2 DCFDA ROS indicator. b Relative mitochondrial ROS levels as measured using MitoSOX mitochondrial superoxide indicator. c – d Relative mitochondrial membrane potential (MMP) as measured using DIOC 6 and JC-1. e Relative expression levels of p-ERK. All the parameters were measured using flow cytometry. Each line represents one donor; n ≥ 3. * p

    Techniques Used: Expressing, Flow Cytometry, Cytometry

    23) Product Images from "Photobiomodulation with 670 nm light increased phagocytosis in human retinal pigment epithelial cells"

    Article Title: Photobiomodulation with 670 nm light increased phagocytosis in human retinal pigment epithelial cells

    Journal: Molecular Vision

    doi:

    Photobiomodulation reduced ROS production but did not change the cell viability or mitochondrial membrane potential. A : Cell viability was measured using a WST-8 assay kit. Cell viability was reduced by oxidative stress, and photobiomodulation did not change the cell viability compared to the H 2 O 2 only group. In this point, we performed the statistical analysis between H 2 O 2 only treated group and photobimodulation group. B : ROS production was measured using the general oxidative stress indicator, CM-H 2 DCFDA. Oxidative stress significantly increased ROS generation, and photobiomodulation reduced ROS production. NAC used as a positive controls also reduced ROS production compared to the H 2 O 2 only group. In this point, we performed the statistical analysis between H 2 O 2 only treated group and photobimodulation group. C : Mitochondrial membrane potential was evaluated using JC-1 dye. Green fluorescence indicates the low mitochondrial membrane potential, and red fluorescence indicates the low mitochondrial membrane potential. Oxidative stress and photobiomodulation did not change the mitochondrial membrane potential. Scale bar=500 μm. The data are expressed as means ± SEM (n=5 or 6) *p
    Figure Legend Snippet: Photobiomodulation reduced ROS production but did not change the cell viability or mitochondrial membrane potential. A : Cell viability was measured using a WST-8 assay kit. Cell viability was reduced by oxidative stress, and photobiomodulation did not change the cell viability compared to the H 2 O 2 only group. In this point, we performed the statistical analysis between H 2 O 2 only treated group and photobimodulation group. B : ROS production was measured using the general oxidative stress indicator, CM-H 2 DCFDA. Oxidative stress significantly increased ROS generation, and photobiomodulation reduced ROS production. NAC used as a positive controls also reduced ROS production compared to the H 2 O 2 only group. In this point, we performed the statistical analysis between H 2 O 2 only treated group and photobimodulation group. C : Mitochondrial membrane potential was evaluated using JC-1 dye. Green fluorescence indicates the low mitochondrial membrane potential, and red fluorescence indicates the low mitochondrial membrane potential. Oxidative stress and photobiomodulation did not change the mitochondrial membrane potential. Scale bar=500 μm. The data are expressed as means ± SEM (n=5 or 6) *p

    Techniques Used: Fluorescence

    24) Product Images from "Experimental Therapy of Ovarian Cancer with Synthetic Makaluvamine Analog: In Vitro and In Vivo Anticancer Activity and Molecular Mechanisms of Action"

    Article Title: Experimental Therapy of Ovarian Cancer with Synthetic Makaluvamine Analog: In Vitro and In Vivo Anticancer Activity and Molecular Mechanisms of Action

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0020729

    FBA-TPQ causes dissipation of the mitochondrial membrane potential and exerts potent tumor growth inhibition. A and B, Fluorescence intensity (JC-1 produces red fluorescence within the mitochondria as JC-1-aggregates while emits green fluorescence when leaks into the cytoplasm as JC-1-monomers; Fluorescence intensity shift between green and red is proportional to the ΔΨm change) values of JC-1 dye at specific excitation wavelengths and the corresponding ratio of Red/Green change (% of the control) after exposure to FBA-TPQ for 24 h (*, p
    Figure Legend Snippet: FBA-TPQ causes dissipation of the mitochondrial membrane potential and exerts potent tumor growth inhibition. A and B, Fluorescence intensity (JC-1 produces red fluorescence within the mitochondria as JC-1-aggregates while emits green fluorescence when leaks into the cytoplasm as JC-1-monomers; Fluorescence intensity shift between green and red is proportional to the ΔΨm change) values of JC-1 dye at specific excitation wavelengths and the corresponding ratio of Red/Green change (% of the control) after exposure to FBA-TPQ for 24 h (*, p

    Techniques Used: Inhibition, Fluorescence

    25) Product Images from "Neuroprotective Effects of Cryptotanshinone in a Direct Reprogramming Model of Parkinson’s Disease"

    Article Title: Neuroprotective Effects of Cryptotanshinone in a Direct Reprogramming Model of Parkinson’s Disease

    Journal: Molecules

    doi: 10.3390/molecules25163602

    Evaluation of the regulation of mitochondrial functions by CTN treatment under MG132-induced oxidative stress. ( A ) MG132-induced increases in mitochondrial reactive oxygen species (ROS) levels and restoration by CTN treatment as analyzed by MitoSOX-based flow cytometry. ( B ) Quantification of mtROS levels. ( C ) Evaluation of mitochondrial membrane potential (MMP) through JC-1 aggregates and JC-1 monomer fluorescence intensity through flow cytometry. ( D ) Quantification of JC-1 aggregates population. ( E ) Evaluation of JC-1 monomers. Data are mean ± standard error of means (SEM) of at least three independent experiments. p -values were analyzed using the unpaired two-tailed Student’s t -test (* p
    Figure Legend Snippet: Evaluation of the regulation of mitochondrial functions by CTN treatment under MG132-induced oxidative stress. ( A ) MG132-induced increases in mitochondrial reactive oxygen species (ROS) levels and restoration by CTN treatment as analyzed by MitoSOX-based flow cytometry. ( B ) Quantification of mtROS levels. ( C ) Evaluation of mitochondrial membrane potential (MMP) through JC-1 aggregates and JC-1 monomer fluorescence intensity through flow cytometry. ( D ) Quantification of JC-1 aggregates population. ( E ) Evaluation of JC-1 monomers. Data are mean ± standard error of means (SEM) of at least three independent experiments. p -values were analyzed using the unpaired two-tailed Student’s t -test (* p

    Techniques Used: Flow Cytometry, Fluorescence, Two Tailed Test

    26) Product Images from "Risuteganib Protects against Hydroquinone–induced Injury in Human RPE Cells"

    Article Title: Risuteganib Protects against Hydroquinone–induced Injury in Human RPE Cells

    Journal: Investigative Ophthalmology & Visual Science

    doi: 10.1167/iovs.61.10.35

    RSG cotreatment protected against the HQ-induced deleterious effect on mitochondrial function. ( A ) RPE cells in triplicate wells of a 96-well plate were treated for 2.5 hours with HQ (150 µM) in the presence or absence of RSG. Cell viability as reflected by mitochondrial dehydrogenase activity was measured with the WST-1 reagent. RSG cotreatment significantly protected cells from HQ-decreased cell viability. Data are representative of three separate experiments with similar results. ( B ) RPE cells in triplicate wells were treated for 1.5 hours with HQ (175 µM) in the presence or absence of RSG. Basal respiration, maximal OCR, ATP production, and spare respiratory capacity were significantly decreased in HQ-treated cells versus control, whereas RSG cotreatment significantly increased mitochondrial metabolic parameters when compared with HQ-treated cells. Data are averaged from three separate experiments ( n = 9 per condition). ( C ) RPE cells in triplicate wells of a 96-well plate were loaded with 20 µM CM-H2DCFDA for 30 minutes and washed twice. Cells were treated with HQ (160 µM) in the presence or absence of RSG. ROS production was measured by relative fluorescence units (RFU) at the indicated times using a fluorescence plate reader. RSG cotreatment significantly decreased HQ-induced ROS generation. Data are representative of three separate experiments with similar results. ( D ) RPE cells in triplicate wells of a 96-well plate were loaded with 10 µM JC-1 dye for 30 minutes and washed twice. Cells were treated with HQ (160 µM) in the presence or absence of RSG. The ΔΨm was measured at the indicated times by the fluorescence of JC-1 monomers and aggregates. RSG cotreatment significantly improved HQ-mediated reduction of ΔΨm. Data are representative of three separate experiments with similar results.
    Figure Legend Snippet: RSG cotreatment protected against the HQ-induced deleterious effect on mitochondrial function. ( A ) RPE cells in triplicate wells of a 96-well plate were treated for 2.5 hours with HQ (150 µM) in the presence or absence of RSG. Cell viability as reflected by mitochondrial dehydrogenase activity was measured with the WST-1 reagent. RSG cotreatment significantly protected cells from HQ-decreased cell viability. Data are representative of three separate experiments with similar results. ( B ) RPE cells in triplicate wells were treated for 1.5 hours with HQ (175 µM) in the presence or absence of RSG. Basal respiration, maximal OCR, ATP production, and spare respiratory capacity were significantly decreased in HQ-treated cells versus control, whereas RSG cotreatment significantly increased mitochondrial metabolic parameters when compared with HQ-treated cells. Data are averaged from three separate experiments ( n = 9 per condition). ( C ) RPE cells in triplicate wells of a 96-well plate were loaded with 20 µM CM-H2DCFDA for 30 minutes and washed twice. Cells were treated with HQ (160 µM) in the presence or absence of RSG. ROS production was measured by relative fluorescence units (RFU) at the indicated times using a fluorescence plate reader. RSG cotreatment significantly decreased HQ-induced ROS generation. Data are representative of three separate experiments with similar results. ( D ) RPE cells in triplicate wells of a 96-well plate were loaded with 10 µM JC-1 dye for 30 minutes and washed twice. Cells were treated with HQ (160 µM) in the presence or absence of RSG. The ΔΨm was measured at the indicated times by the fluorescence of JC-1 monomers and aggregates. RSG cotreatment significantly improved HQ-mediated reduction of ΔΨm. Data are representative of three separate experiments with similar results.

    Techniques Used: Activity Assay, Fluorescence

    27) Product Images from "Overexpression of human kynurenine-3-monooxygenase protects against 3-hydroxykynurenine-mediated apoptosis through bidirectional nonlinear feedback"

    Article Title: Overexpression of human kynurenine-3-monooxygenase protects against 3-hydroxykynurenine-mediated apoptosis through bidirectional nonlinear feedback

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2016.87

    KMO protects against 3HK-induced cell death. Graphs showing ( a ) JC-1 readout, n =6 for each cell type and conditions, red:green ratio is significantly decreased ( P =0.0498) in wild-type cells incubated with 3HK compared with KMO cells, ( b ) LDH assay readout, n =6 for each cell type and conditions, LDH release is significantly increased ( P =0.0016) in wild-type cells incubated with 3HK compared with KMO cells, ( c ) caspase 3/7 activity readout, n =6 for each cell type and conditions, caspase activity is increased in wild-type cells incubated with 3HK compared with KMO cells. The S.E.M. is indicated by error bars on all graphs. Data are representative of three independent experiments for each of these assays. ( d ) Heat maps indicating DiOC6 staining in KMO cells verses wild-type cells with a 3HK-dose and time response in the presence and absence of Z-VAD-FMK, data are mean values of n =2 for each cell type and conditions from one independent experiment
    Figure Legend Snippet: KMO protects against 3HK-induced cell death. Graphs showing ( a ) JC-1 readout, n =6 for each cell type and conditions, red:green ratio is significantly decreased ( P =0.0498) in wild-type cells incubated with 3HK compared with KMO cells, ( b ) LDH assay readout, n =6 for each cell type and conditions, LDH release is significantly increased ( P =0.0016) in wild-type cells incubated with 3HK compared with KMO cells, ( c ) caspase 3/7 activity readout, n =6 for each cell type and conditions, caspase activity is increased in wild-type cells incubated with 3HK compared with KMO cells. The S.E.M. is indicated by error bars on all graphs. Data are representative of three independent experiments for each of these assays. ( d ) Heat maps indicating DiOC6 staining in KMO cells verses wild-type cells with a 3HK-dose and time response in the presence and absence of Z-VAD-FMK, data are mean values of n =2 for each cell type and conditions from one independent experiment

    Techniques Used: Incubation, Lactate Dehydrogenase Assay, Activity Assay, Staining

    28) Product Images from "Subcellular compartmentalization of GPX1 allelic isoforms differentially impact parameters of energy metabolism"

    Article Title: Subcellular compartmentalization of GPX1 allelic isoforms differentially impact parameters of energy metabolism

    Journal: Journal of cellular biochemistry

    doi: 10.1002/jcb.27610

    The effect of GPX1 isoforms on mitochondrial membrane potential. The mitochondrial membrane potential of transfected cells was determined using JC-1 fluorescence detection described in the Methods. The data is the ratio of red fluorescence (595 nm) which is proportional to the membrane potential and the green fluorescence (535 nm) which is emitted in necrotic or apoptotic cells. The statistical significances were calculated from the similarly treated vector transfected control and the values are the means ± sd, n = 3. ** P
    Figure Legend Snippet: The effect of GPX1 isoforms on mitochondrial membrane potential. The mitochondrial membrane potential of transfected cells was determined using JC-1 fluorescence detection described in the Methods. The data is the ratio of red fluorescence (595 nm) which is proportional to the membrane potential and the green fluorescence (535 nm) which is emitted in necrotic or apoptotic cells. The statistical significances were calculated from the similarly treated vector transfected control and the values are the means ± sd, n = 3. ** P

    Techniques Used: Transfection, Fluorescence, Plasmid Preparation

    29) Product Images from "The E3 ligase Mule protects the heart against oxidative stress and mitochondrial dysfunction through Myc-dependent inactivation of Pgc-1α and Pink1"

    Article Title: The E3 ligase Mule protects the heart against oxidative stress and mitochondrial dysfunction through Myc-dependent inactivation of Pgc-1α and Pink1

    Journal: Scientific Reports

    doi: 10.1038/srep41490

    Enhanced oxidative stress in mice with cardiac-specific deletion of Mule. ( A ) Immunoblot analysis of factors involved in detoxification processes of reactive oxygen species in left ventricular extracts of Mule fl / fl ( y ) ;mcm and DKO mice at 7 days post-Tam employing specific antibodies as indicated on the left. One representative result of three independent experiments is shown. ( B ) The membrane potential (ΔΨm) of mitochondria isolated from Mule mutant mice is susceptible to ROS-induced depolarization. Genetic co-ablation of Myc and Mule in DKO mice rescues antimycin induced decreases of ΔΨm in mitochondria derived from this double mutant strain. Isolated mitochondria, incubated with JC-1 (5 μg/mL), were treated with antimycin (50 μM). JC-1 emission at 535/595 nm was recorded at 1 reading/min for 30 min using a fluorescence spectrophotometer. The rate between two time points (Δemission at 595 nm/min) was calculated in the most linear range of decline for JC-1 fluorescence intensity. n = 4. ( C , D ) Mitochondrial aconitase ( C ) and catalase ( D ) activities were determined spectrophotometrically in ventricular samples from Mule fl / fl ( y ) ;mcm and DKO strains at 7 days post-Tam. n = 4. ( E ) Oxidative genomic DNA damage in the hearts of Mule fl / fl ( y ) ;mcm mice is abolished by genetic co-ablation of Myc in DKO animals. Concentrations of 8-hydroxy-2′-deoxyguanosine (8- OHdG), a biomarker for oxidative DNA damage in the indicated strains was determined by a competitive enzyme-linked ELISA employing 8-OHdG antibodies. n = 4. ( F ) Higher levels of 4-hydroxyalkenals (4-HAE), an indicator of ROS-dependent lipid peroxidation in ventricular extracts of Mule fl / fl ( y ) ;mcm compared with DKO. n = 4. ( G ) Significantly reduced levels of NADPH, an essential cofactor for the reduction of glutathione, in in ventricul*ar extracts of Mule fl / fl ( y ) ;mcm mice versus DKO. n = 4. ( H , I ) Decreased the reduced glutathione/oxidized glutathione (GSH/GSSG) ratios, an indicator of cardiac oxidative stress, in ventricular samples from Mule fl / fl ( y ) ;mcm mice that was prevented by Myc co-ablation in DKO. n = 4. Schematic model for Mule-mediated inhibition of Myc-dependent cardiac hypertrophy. In response to genetic ablation of Mule, Myc is activated and transcriptionally inhibits canonical downstream targets Pgc-1α and Pink1 promoting the development of heart failure by induction of oxidative stress and mitochondrial dysfunction. Data are means ± s.e.m.
    Figure Legend Snippet: Enhanced oxidative stress in mice with cardiac-specific deletion of Mule. ( A ) Immunoblot analysis of factors involved in detoxification processes of reactive oxygen species in left ventricular extracts of Mule fl / fl ( y ) ;mcm and DKO mice at 7 days post-Tam employing specific antibodies as indicated on the left. One representative result of three independent experiments is shown. ( B ) The membrane potential (ΔΨm) of mitochondria isolated from Mule mutant mice is susceptible to ROS-induced depolarization. Genetic co-ablation of Myc and Mule in DKO mice rescues antimycin induced decreases of ΔΨm in mitochondria derived from this double mutant strain. Isolated mitochondria, incubated with JC-1 (5 μg/mL), were treated with antimycin (50 μM). JC-1 emission at 535/595 nm was recorded at 1 reading/min for 30 min using a fluorescence spectrophotometer. The rate between two time points (Δemission at 595 nm/min) was calculated in the most linear range of decline for JC-1 fluorescence intensity. n = 4. ( C , D ) Mitochondrial aconitase ( C ) and catalase ( D ) activities were determined spectrophotometrically in ventricular samples from Mule fl / fl ( y ) ;mcm and DKO strains at 7 days post-Tam. n = 4. ( E ) Oxidative genomic DNA damage in the hearts of Mule fl / fl ( y ) ;mcm mice is abolished by genetic co-ablation of Myc in DKO animals. Concentrations of 8-hydroxy-2′-deoxyguanosine (8- OHdG), a biomarker for oxidative DNA damage in the indicated strains was determined by a competitive enzyme-linked ELISA employing 8-OHdG antibodies. n = 4. ( F ) Higher levels of 4-hydroxyalkenals (4-HAE), an indicator of ROS-dependent lipid peroxidation in ventricular extracts of Mule fl / fl ( y ) ;mcm compared with DKO. n = 4. ( G ) Significantly reduced levels of NADPH, an essential cofactor for the reduction of glutathione, in in ventricul*ar extracts of Mule fl / fl ( y ) ;mcm mice versus DKO. n = 4. ( H , I ) Decreased the reduced glutathione/oxidized glutathione (GSH/GSSG) ratios, an indicator of cardiac oxidative stress, in ventricular samples from Mule fl / fl ( y ) ;mcm mice that was prevented by Myc co-ablation in DKO. n = 4. Schematic model for Mule-mediated inhibition of Myc-dependent cardiac hypertrophy. In response to genetic ablation of Mule, Myc is activated and transcriptionally inhibits canonical downstream targets Pgc-1α and Pink1 promoting the development of heart failure by induction of oxidative stress and mitochondrial dysfunction. Data are means ± s.e.m.

    Techniques Used: Mouse Assay, Isolation, Mutagenesis, Derivative Assay, Incubation, Fluorescence, Spectrophotometry, Biomarker Assay, Enzyme-linked Immunosorbent Assay, Inhibition, Pyrolysis Gas Chromatography

    30) Product Images from "Evaluation of the Cytotoxicity and Genotoxicity of Flavonolignans in Different Cellular Models"

    Article Title: Evaluation of the Cytotoxicity and Genotoxicity of Flavonolignans in Different Cellular Models

    Journal: Nutrients

    doi: 10.3390/nu9121356

    The effect of flavonolignans (silychristin, silybin and silydianin in concentrations of 10, 50 and 100 µM) on blood platelet mitochondrial membrane potential. MMP is expressed as a ratio of 530 nm/590 nm to 485 nm/538 nm (aggregates to monomer) fluorescence, as quantified with a fluorescent plate reader after JC-1 staining. The data represent means of ± standard deviation (SD), n = 12, * p
    Figure Legend Snippet: The effect of flavonolignans (silychristin, silybin and silydianin in concentrations of 10, 50 and 100 µM) on blood platelet mitochondrial membrane potential. MMP is expressed as a ratio of 530 nm/590 nm to 485 nm/538 nm (aggregates to monomer) fluorescence, as quantified with a fluorescent plate reader after JC-1 staining. The data represent means of ± standard deviation (SD), n = 12, * p

    Techniques Used: Fluorescence, Staining, Standard Deviation

    31) Product Images from "Highly Active Antiretroviral Therapy Drug Combination Induces Oxidative Stress and Mitochondrial Dysfunction in Immortalized Human Blood-Brain Barrier Endothelial Cells"

    Article Title: Highly Active Antiretroviral Therapy Drug Combination Induces Oxidative Stress and Mitochondrial Dysfunction in Immortalized Human Blood-Brain Barrier Endothelial Cells

    Journal: Free radical biology & medicine

    doi: 10.1016/j.freeradbiomed.2010.12.029

    Analysis of mitochondrial membrane potential. ΔΨm was analyzed in adherent hCMEC/D3 after 72 hr of AZT+IDV treatment, using the potential-dependent aggregate-forming lipophilic cation JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazole carbocyanide iodide). Left image represents JC-1 dye aggregation in mitochondria with intact membrane potential. Mitochondria exhibit red fluorescence if ΔΨm is preserved. Middle image represents JC-1 monomers exhibiting green fluorescence in cytoplasm of cells. Mitochondria in treated cells showed a dose-dependent decrease in red fluorescence due to loss of ΔΨm. Merged images are represented on extreme right.
    Figure Legend Snippet: Analysis of mitochondrial membrane potential. ΔΨm was analyzed in adherent hCMEC/D3 after 72 hr of AZT+IDV treatment, using the potential-dependent aggregate-forming lipophilic cation JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazole carbocyanide iodide). Left image represents JC-1 dye aggregation in mitochondria with intact membrane potential. Mitochondria exhibit red fluorescence if ΔΨm is preserved. Middle image represents JC-1 monomers exhibiting green fluorescence in cytoplasm of cells. Mitochondria in treated cells showed a dose-dependent decrease in red fluorescence due to loss of ΔΨm. Merged images are represented on extreme right.

    Techniques Used: Fluorescence

    32) Product Images from "Toward Highly Potent Cancer Agents by Modulating the C-2 Group of the Arylthioindole Class of Tubulin Polymerization Inhibitors"

    Article Title: Toward Highly Potent Cancer Agents by Modulating the C-2 Group of the Arylthioindole Class of Tubulin Polymerization Inhibitors

    Journal: Journal of medicinal chemistry

    doi: 10.1021/jm3013097

    Effects of a 24 h treatment with 100 nM 57 or 18 or 30 μ M CCCP on mitochondrial transmembrane potential as assessed by JC-1 staining. Graphs indicate the ΔΨdissipation expressed as red/green (R/G) fluorescence ratio. Each value has been normalized versus the R/G ratio of the vehicle treated control to which an arbitrary value of 100% has been assigned. Data were the mean of two independent experiments performed in triplicate ((**) p
    Figure Legend Snippet: Effects of a 24 h treatment with 100 nM 57 or 18 or 30 μ M CCCP on mitochondrial transmembrane potential as assessed by JC-1 staining. Graphs indicate the ΔΨdissipation expressed as red/green (R/G) fluorescence ratio. Each value has been normalized versus the R/G ratio of the vehicle treated control to which an arbitrary value of 100% has been assigned. Data were the mean of two independent experiments performed in triplicate ((**) p

    Techniques Used: Staining, Fluorescence

    33) Product Images from "Impaired cardiac energy metabolism in embryos lacking adrenergic stimulation"

    Article Title: Impaired cardiac energy metabolism in embryos lacking adrenergic stimulation

    Journal: American Journal of Physiology - Endocrinology and Metabolism

    doi: 10.1152/ajpendo.00267.2014

    Analysis of mitochondrial membrane potentials in adrenergic-deficient and control myocytes using flow cytometry and fluorescence microscopy with JC-1 dye. A and C : representative scatter plots of flow cytometry with red and green fluorescence from JC-1 dye in adrenergic-deficient and adrenergic-competent E10.5 and E11.5 embryonic primary cardiomyocytes. Adrenergic-competent samples (red) and adrenergic-deficient samples (blue) in histogram overlay. B and D : ratio of red/green fluorescence in E10.5 and E11.5 adrenergic-competent (open bars) and -deficient (black bars) samples. Numerical values within the bars refer to the no. ( n ) of samples analyzed. E : representative scanning laser confocal microscopy of JC-1 dye in E11.5 primary cardiomyocytes. Scale bar, 10 μm.
    Figure Legend Snippet: Analysis of mitochondrial membrane potentials in adrenergic-deficient and control myocytes using flow cytometry and fluorescence microscopy with JC-1 dye. A and C : representative scatter plots of flow cytometry with red and green fluorescence from JC-1 dye in adrenergic-deficient and adrenergic-competent E10.5 and E11.5 embryonic primary cardiomyocytes. Adrenergic-competent samples (red) and adrenergic-deficient samples (blue) in histogram overlay. B and D : ratio of red/green fluorescence in E10.5 and E11.5 adrenergic-competent (open bars) and -deficient (black bars) samples. Numerical values within the bars refer to the no. ( n ) of samples analyzed. E : representative scanning laser confocal microscopy of JC-1 dye in E11.5 primary cardiomyocytes. Scale bar, 10 μm.

    Techniques Used: Flow Cytometry, Cytometry, Fluorescence, Microscopy, Confocal Microscopy

    34) Product Images from "Jacaric acid inhibits the growth of murine macrophage-like leukemia PU5-1.8 cells by inducing cell cycle arrest and apoptosis"

    Article Title: Jacaric acid inhibits the growth of murine macrophage-like leukemia PU5-1.8 cells by inducing cell cycle arrest and apoptosis

    Journal: Cancer Cell International

    doi: 10.1186/s12935-015-0246-5

    Jacaric acid induces mitochondrial membrane depolarization in PU5-1.8 cells. PU5-1.8 cells were incubated with either ethanol control ( a ), or 4 μM jacaric acid ( b ), 8 μM jacaric acid ( c ) and 12 μM jacaric acid ( d ) at 37 °C for 24 h. After incubation, the cells were stained with JC-1 dye. The fluorescence intensity was measured by the FACSCanto™ flow cytometer. e The results were quantified and expressed as mean ± SE. *** p
    Figure Legend Snippet: Jacaric acid induces mitochondrial membrane depolarization in PU5-1.8 cells. PU5-1.8 cells were incubated with either ethanol control ( a ), or 4 μM jacaric acid ( b ), 8 μM jacaric acid ( c ) and 12 μM jacaric acid ( d ) at 37 °C for 24 h. After incubation, the cells were stained with JC-1 dye. The fluorescence intensity was measured by the FACSCanto™ flow cytometer. e The results were quantified and expressed as mean ± SE. *** p

    Techniques Used: Incubation, Staining, Fluorescence, Flow Cytometry, Cytometry

    35) Product Images from "Physiological Effects of Ac4ManNAz and Optimization of Metabolic Labeling for Cell Tracking"

    Article Title: Physiological Effects of Ac4ManNAz and Optimization of Metabolic Labeling for Cell Tracking

    Journal: Theranostics

    doi: 10.7150/thno.17711

    Change of Mitochondria function in A549 cells treated with Ac4ManNAz. (A) Mitochondrial membrane potential (ΔΨm) was measured in A549 cells by JC-1 staining as described in materials and methods. (B) A representative graph of ECAR outputs from the XF96 analyzer of the control, and Ac4ManNAz treated A549 cells and the response to glucose, oligomycin and 2-deoxy-glucose. (C) A representative graph of OCR outputs from the XF96 analyzer of the control, and Ac4ManNAz treated A549 cells and the response to oligomycin, FCCP, and antimycin A/rotenone.
    Figure Legend Snippet: Change of Mitochondria function in A549 cells treated with Ac4ManNAz. (A) Mitochondrial membrane potential (ΔΨm) was measured in A549 cells by JC-1 staining as described in materials and methods. (B) A representative graph of ECAR outputs from the XF96 analyzer of the control, and Ac4ManNAz treated A549 cells and the response to glucose, oligomycin and 2-deoxy-glucose. (C) A representative graph of OCR outputs from the XF96 analyzer of the control, and Ac4ManNAz treated A549 cells and the response to oligomycin, FCCP, and antimycin A/rotenone.

    Techniques Used: Staining

    36) Product Images from "4′‐Phosphopantetheine corrects CoA, iron, and dopamine metabolic defects in mammalian models of PKAN"

    Article Title: 4′‐Phosphopantetheine corrects CoA, iron, and dopamine metabolic defects in mammalian models of PKAN

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201910489

    Human cells with PANK 2 mutations show perturbations in the CoA synthesis pathway and diminished mitochondrial respiration Relative quantification of COASY mRNA in human PKAN primary cell lines. n = 5 for both genotypes and cell types. Relative quantification of COASY / Coasy in fresh, blood‐derived lymphocytes from human and mouse, respectively compared by genotype. n = 51, 35 for human (control, PKAN, respectively) and n = 5 for both mouse genotypes. Extracellular flux analysis in human fibroblasts showing differences in OCR (oxygen consumption rate) and ECAR (extracellular acidification rate) by genotype. Oligom; oligomycin, FCCP; carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone, 2‐DG; 2‐deoxy glucose. Quantification of baseline OCR and ECAR is shown, as well. n = 3 per genotype with four technical replicates. Complex I activity assay using fibroblast lysates. Cells were either treated with vehicle or 50 μM pPanSH for 3 days, and total protein extract was used for the Complex I Dipstick assay n = 4 for both genotypes and treatment groups. Relative mRNA expression of mitochondrially encoded genes important for oxidative phosphorylation from complex I ( MT‐ND1 ), complex III ( MT‐CYB ), cytochrome C ( MT‐CO1 ), and ATP synthase ( MT‐ATP6 ) in human mutant fibroblasts versus control cells. n = 3 for both genotypes. Calculation of active mitochondria using the JC‐1 assay by genotype in control and PKAN lymphoblasts. n = 2. Data information: Data were analyzed using one‐way or two‐way ANOVA for statistical significance. * P
    Figure Legend Snippet: Human cells with PANK 2 mutations show perturbations in the CoA synthesis pathway and diminished mitochondrial respiration Relative quantification of COASY mRNA in human PKAN primary cell lines. n = 5 for both genotypes and cell types. Relative quantification of COASY / Coasy in fresh, blood‐derived lymphocytes from human and mouse, respectively compared by genotype. n = 51, 35 for human (control, PKAN, respectively) and n = 5 for both mouse genotypes. Extracellular flux analysis in human fibroblasts showing differences in OCR (oxygen consumption rate) and ECAR (extracellular acidification rate) by genotype. Oligom; oligomycin, FCCP; carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone, 2‐DG; 2‐deoxy glucose. Quantification of baseline OCR and ECAR is shown, as well. n = 3 per genotype with four technical replicates. Complex I activity assay using fibroblast lysates. Cells were either treated with vehicle or 50 μM pPanSH for 3 days, and total protein extract was used for the Complex I Dipstick assay n = 4 for both genotypes and treatment groups. Relative mRNA expression of mitochondrially encoded genes important for oxidative phosphorylation from complex I ( MT‐ND1 ), complex III ( MT‐CYB ), cytochrome C ( MT‐CO1 ), and ATP synthase ( MT‐ATP6 ) in human mutant fibroblasts versus control cells. n = 3 for both genotypes. Calculation of active mitochondria using the JC‐1 assay by genotype in control and PKAN lymphoblasts. n = 2. Data information: Data were analyzed using one‐way or two‐way ANOVA for statistical significance. * P

    Techniques Used: Derivative Assay, Activity Assay, Expressing, Mutagenesis

    37) Product Images from "Defects in calcium homeostasis and mitochondria can be reversed in Pompe disease"

    Article Title: Defects in calcium homeostasis and mitochondria can be reversed in Pompe disease

    Journal: Autophagy

    doi: 10.1080/15548627.2015.1009779

    Evaluation of ΔΨ m , using JC-1 dye, in myotubes from Pompe patients. Human primary myoblasts were derived from a healthy individual (control; N) and 2 Pompe patients with the adult form of the disease (P#484 P#542). Cultured myotubes
    Figure Legend Snippet: Evaluation of ΔΨ m , using JC-1 dye, in myotubes from Pompe patients. Human primary myoblasts were derived from a healthy individual (control; N) and 2 Pompe patients with the adult form of the disease (P#484 P#542). Cultured myotubes

    Techniques Used: Derivative Assay, Cell Culture

    Changes in mitochondrial physiological parameters in KO myotubes. WT and KO myotubes were analyzed on d 7 in differentiation medium. ( A ) Green JC-1 dye aggregates and becomes red when ΔΨ m is preserved (top). There is a significant degree
    Figure Legend Snippet: Changes in mitochondrial physiological parameters in KO myotubes. WT and KO myotubes were analyzed on d 7 in differentiation medium. ( A ) Green JC-1 dye aggregates and becomes red when ΔΨ m is preserved (top). There is a significant degree

    Techniques Used:

    38) Product Images from "Para-Phenylenediamine Induces Apoptotic Death of Melanoma Cells and Reduces Melanoma Tumour Growth in Mice"

    Article Title: Para-Phenylenediamine Induces Apoptotic Death of Melanoma Cells and Reduces Melanoma Tumour Growth in Mice

    Journal: Biochemistry Research International

    doi: 10.1155/2016/3137010

    p-PD depolarizes mitochondrial membrane potential of both melanoma cells. p-PD treated (as indicated for two hours) and untreated (0 μ g/mL) melanoma cells were subjected to JC 1 staining for the measurement of MMP. For both cell lines, cell clusters which are high in PE fluorescence are gated in the top of each dot plot (red and green for B16-F10 and A375 cells, resp.) presenting the healthy cell population. Cell clusters that are gated for low PE fluorescence in the bottom part of each dot plot (green and blue for B16-F10 and A375 cells, resp.) are representing the cells with depolarized mitochondria. Cell percentage values of the gated population are stating the effect of p-PD in the polarization of mitochondria. One piece of the representative experimental data is shown here. Each set was performed in triplicate.
    Figure Legend Snippet: p-PD depolarizes mitochondrial membrane potential of both melanoma cells. p-PD treated (as indicated for two hours) and untreated (0 μ g/mL) melanoma cells were subjected to JC 1 staining for the measurement of MMP. For both cell lines, cell clusters which are high in PE fluorescence are gated in the top of each dot plot (red and green for B16-F10 and A375 cells, resp.) presenting the healthy cell population. Cell clusters that are gated for low PE fluorescence in the bottom part of each dot plot (green and blue for B16-F10 and A375 cells, resp.) are representing the cells with depolarized mitochondria. Cell percentage values of the gated population are stating the effect of p-PD in the polarization of mitochondria. One piece of the representative experimental data is shown here. Each set was performed in triplicate.

    Techniques Used: Staining, Fluorescence

    39) Product Images from "Shenqi Fuzheng Injection Reverses Cisplatin Resistance through Mitofusin-2-Mediated Cell Cycle Arrest and Apoptosis in A549/DDP Cells"

    Article Title: Shenqi Fuzheng Injection Reverses Cisplatin Resistance through Mitofusin-2-Mediated Cell Cycle Arrest and Apoptosis in A549/DDP Cells

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2018/8258246

    Mfn2 is involved in reversing cell cycle inhibition and cell apoptosis upon cotreatment with cisplatin and SFI. (a, b, and c) A549/DDP cells were pretreated with various concentrations (2, 3.78, and 35.18 mg/mL) of SFI for 2 hours and then exposed to cisplatin (40 μ g/mL) for another 24 hours. After drug intervention, (a) cell lysates were prepared and subjected to immunoblotting with antibodies to Mfn2 and β -actin; (b) cells were incubated with JC-1 and analyzed by flow cytometry; and (c) cells were labeled with DCFH-DA and the fluorescence intensity of the oxidized product DCF in individual cells was detected by flow cytometry and fluorescence microscopy. (d, e, and f) A549/DDP cells were pretreated with or without 2.5 mM of NAC, followed by cisplatin (40 μ g/mL) and SFI (35.18 mg/mL) cotreatment. After drug intervention, (d) cell lysates were prepared and subjected to immunoblotting with antibodies to Mfn2 and β -actin; (e) cell cycle distribution by PI staining and DNA contents were determined by flow cytometry; and (f) apoptosis was determined by Annexin V-FITC/PI staining and analyzed by flow cytometry.
    Figure Legend Snippet: Mfn2 is involved in reversing cell cycle inhibition and cell apoptosis upon cotreatment with cisplatin and SFI. (a, b, and c) A549/DDP cells were pretreated with various concentrations (2, 3.78, and 35.18 mg/mL) of SFI for 2 hours and then exposed to cisplatin (40 μ g/mL) for another 24 hours. After drug intervention, (a) cell lysates were prepared and subjected to immunoblotting with antibodies to Mfn2 and β -actin; (b) cells were incubated with JC-1 and analyzed by flow cytometry; and (c) cells were labeled with DCFH-DA and the fluorescence intensity of the oxidized product DCF in individual cells was detected by flow cytometry and fluorescence microscopy. (d, e, and f) A549/DDP cells were pretreated with or without 2.5 mM of NAC, followed by cisplatin (40 μ g/mL) and SFI (35.18 mg/mL) cotreatment. After drug intervention, (d) cell lysates were prepared and subjected to immunoblotting with antibodies to Mfn2 and β -actin; (e) cell cycle distribution by PI staining and DNA contents were determined by flow cytometry; and (f) apoptosis was determined by Annexin V-FITC/PI staining and analyzed by flow cytometry.

    Techniques Used: Inhibition, Incubation, Flow Cytometry, Cytometry, Labeling, Fluorescence, Microscopy, Staining

    40) Product Images from "Hyperoside alleviates adriamycin-induced podocyte injury via inhibiting mitochondrial fission"

    Article Title: Hyperoside alleviates adriamycin-induced podocyte injury via inhibiting mitochondrial fission

    Journal: Oncotarget

    doi: 10.18632/oncotarget.21287

    Effect of hyperoside on adriamycin-induced mitochondrial dysfunction in vitro Podocytes were pre-treated with hyperoside (50μmol/L) for 1 h followed by co-incubation with ADR (1μg/ml) for further 12 h. (A) Western blots for PGC-1α expression. Left: representative immunoblots. Right: densitometric analysis. (B) mtDNA copy number. (C) Representative images of podocytes stained with MitoSOX. (D) Quantitation of MitoSOX by flow cytometry. (E) Podocytes were stained with DCFDA and the DCF fluorescence intensities were analyzed by flow cytometry. (F) Representative images of podocytes stained with JC-1. (G) Quantitation of mitochondrial membrane potential changes by flow cytometry. Values are means ± SEM from three independent experiments. * P
    Figure Legend Snippet: Effect of hyperoside on adriamycin-induced mitochondrial dysfunction in vitro Podocytes were pre-treated with hyperoside (50μmol/L) for 1 h followed by co-incubation with ADR (1μg/ml) for further 12 h. (A) Western blots for PGC-1α expression. Left: representative immunoblots. Right: densitometric analysis. (B) mtDNA copy number. (C) Representative images of podocytes stained with MitoSOX. (D) Quantitation of MitoSOX by flow cytometry. (E) Podocytes were stained with DCFDA and the DCF fluorescence intensities were analyzed by flow cytometry. (F) Representative images of podocytes stained with JC-1. (G) Quantitation of mitochondrial membrane potential changes by flow cytometry. Values are means ± SEM from three independent experiments. * P

    Techniques Used: In Vitro, Incubation, Western Blot, Pyrolysis Gas Chromatography, Expressing, Staining, Quantitation Assay, Flow Cytometry, Cytometry, Fluorescence

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    Staining:

    Article Title: Unique properties of a subset of human pluripotent stem cells with high capacity for self-renewal
    Article Snippet: .. 1 × 106 single cells that were stained with antibodies were resuspended in 1 mL of mTeSR1 supplemented with 250 nM TMRM (Thermo Fisher, Cat. #T668) or 0.3 μg/mL JC-1 (Thermo Fisher, Cat. #T3168) and incubated in the incubator for 30 min. .. The media was subsequently removed and the cells were washed three times with mTeSR1 before analysis on the flow cytometer.

    Article Title: Combination of Sulindac and Dichloroacetate Kills Cancer Cells via Oxidative Damage
    Article Snippet: .. JC-1 Staining to Monitor Mitochondrial Membrane Potential Mitochondrial membrane potential was determined using the JC-1 dye (Molecular Probes). ..

    Article Title: Tumor‐stroma interactions differentially alter drug sensitivity based on the origin of stromal cells
    Article Snippet: .. 1,500 stained cells were plated in 40 μl FluoroBrite media (Thermofisher # A1896701), supplemented with 10% FBS, 2 mM glutamine, and penicillin/streptomycin, in a Greiner clear 384‐well plate (#781986) and allowed to adhere for 3 h. Cancer cell lines were then trypsinized and stained with 1.5 μg/ml (final concentration) JC‐1 (Thermofisher # T3168) in FluoroBrite at a concentration of 1 × 106 cell/ml for 20 min at 37°C. ..

    Incubation:

    Article Title: Unique properties of a subset of human pluripotent stem cells with high capacity for self-renewal
    Article Snippet: .. 1 × 106 single cells that were stained with antibodies were resuspended in 1 mL of mTeSR1 supplemented with 250 nM TMRM (Thermo Fisher, Cat. #T668) or 0.3 μg/mL JC-1 (Thermo Fisher, Cat. #T3168) and incubated in the incubator for 30 min. .. The media was subsequently removed and the cells were washed three times with mTeSR1 before analysis on the flow cytometer.

    Article Title: Topical Application of the Antimicrobial Agent Triclosan Induces NLRP3 Inflammasome Activation and Mitochondrial Dysfunction
    Article Snippet: .. Briefly, dLN single-cell suspensions were diluted to 1 × 106 cells/ml with a final concentration of 2μM JC-1 dye in triclosan-free PBS and incubated for 15 min at 37°C/5% CO2. .. CCCP was used as a positive control.

    Imaging:

    Article Title: Cumulative mitochondrial activity correlates with ototoxin susceptibility in zebrafish mechanosensory hair cells
    Article Snippet: .. The same imaging and analysis protocol was followed for static imaging of zebrafish labeled with JC-1 dye (ThermoFisher). .. To load the dye, free-swimming larvae were incubated in 1.5 μM JC-1 (diluted in embryo media) for 30 min and then washed three times in embryo media.

    Labeling:

    Article Title: Cumulative mitochondrial activity correlates with ototoxin susceptibility in zebrafish mechanosensory hair cells
    Article Snippet: .. The same imaging and analysis protocol was followed for static imaging of zebrafish labeled with JC-1 dye (ThermoFisher). .. To load the dye, free-swimming larvae were incubated in 1.5 μM JC-1 (diluted in embryo media) for 30 min and then washed three times in embryo media.

    Concentration Assay:

    Article Title: Tumor‐stroma interactions differentially alter drug sensitivity based on the origin of stromal cells
    Article Snippet: .. 1,500 stained cells were plated in 40 μl FluoroBrite media (Thermofisher # A1896701), supplemented with 10% FBS, 2 mM glutamine, and penicillin/streptomycin, in a Greiner clear 384‐well plate (#781986) and allowed to adhere for 3 h. Cancer cell lines were then trypsinized and stained with 1.5 μg/ml (final concentration) JC‐1 (Thermofisher # T3168) in FluoroBrite at a concentration of 1 × 106 cell/ml for 20 min at 37°C. ..

    Article Title: Topical Application of the Antimicrobial Agent Triclosan Induces NLRP3 Inflammasome Activation and Mitochondrial Dysfunction
    Article Snippet: .. Briefly, dLN single-cell suspensions were diluted to 1 × 106 cells/ml with a final concentration of 2μM JC-1 dye in triclosan-free PBS and incubated for 15 min at 37°C/5% CO2. .. CCCP was used as a positive control.

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    Thermo Fisher jc 1 dye
    Coculture screen to identify tumor–stroma interactions reveals divergent interactions between fibroblasts and TNBC cells Schematic of screen design. TNBC cell lines were labeled with <t>JC‐1,</t> grown in monoculture or in coculture with primary fibroblast cells, and treated with one of 42 anticancer drugs. JC‐1 fluorescence was monitored using a fluorescence plate reader at 8‐hour intervals for 72 h. Representative images of BT‐20 cells (BL subtype) cocultured with HADF fibroblasts. BT‐20 cells labeled with JC‐1 dye; HADF labeled with a blue cell dye (CellTrace). Images taken before drug addition (B) or 96 h after exposure to 0.5 μM camptothecin (C). Kinetic trace of JC‐1 red fluorescence following exposure to camptothecin as in (B and C). Data are relative JC‐1 red fluorescence, normalized to the well's average prior to drug addition. Data represent mean ± standard deviation for five biological replicates. Total coculture screen data. Each blue dot represents a unique TNBC–fibroblast–drug measurement. 312,120 total measurements of drug response. Orange and purple dots highlight conditions validated in panels (F–I). Orange are HCC‐1143 (BL) cells cocultured with HCPF and treated with palbociclib. Purple dots are Hs578T (ML) cocultured with WS1 and exposed to etoposide. For colored dots, increasing size represents longer drug exposure times. Validation of coculture screening data. (F) Flow cytometry analysis of cell death using Live/Dead Blue stain. FarRed CellTrace was used to label and distinguish fibroblasts. (G) Quantification of percent TNBC cell death in experiment described in panel (F). (H and I) Flow cytometry and quantitative analysis, as in panels (F and G) for Hs578T +/− WS1 and treated with etoposide. Error bars represent standard deviation of biological replicates. P ‐value calculated using t ‐test. Data are from three biological replicates and error bars. *** P ‐value
    Jc 1 Dye, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 175 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TET enhanced the pro-apoptotic effect of PTX in SKOV3/PTX cells. SKOV3/PTX cells were treated with PTX (1 μM) or/and 2 μM TET for 72 h. ( A, B ) Apoptotic cells were detected by Annexin V/PI double staining and flow cytometry in SKOV3/PTX cells. ( C ) <t>JC-1</t> staining was used to determine MMP loss. ( D ) Expression levels of Bax, Bcl-2 and cleaved caspase 3 in SKOV3/PTX cells were detected with Western blotting. ( E – G ) The relative expressions of Bax, Bcl-2 and cleaved caspase 3 were quantified via normalization to β-actin. *P
    Jc 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 398 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Coculture screen to identify tumor–stroma interactions reveals divergent interactions between fibroblasts and TNBC cells Schematic of screen design. TNBC cell lines were labeled with JC‐1, grown in monoculture or in coculture with primary fibroblast cells, and treated with one of 42 anticancer drugs. JC‐1 fluorescence was monitored using a fluorescence plate reader at 8‐hour intervals for 72 h. Representative images of BT‐20 cells (BL subtype) cocultured with HADF fibroblasts. BT‐20 cells labeled with JC‐1 dye; HADF labeled with a blue cell dye (CellTrace). Images taken before drug addition (B) or 96 h after exposure to 0.5 μM camptothecin (C). Kinetic trace of JC‐1 red fluorescence following exposure to camptothecin as in (B and C). Data are relative JC‐1 red fluorescence, normalized to the well's average prior to drug addition. Data represent mean ± standard deviation for five biological replicates. Total coculture screen data. Each blue dot represents a unique TNBC–fibroblast–drug measurement. 312,120 total measurements of drug response. Orange and purple dots highlight conditions validated in panels (F–I). Orange are HCC‐1143 (BL) cells cocultured with HCPF and treated with palbociclib. Purple dots are Hs578T (ML) cocultured with WS1 and exposed to etoposide. For colored dots, increasing size represents longer drug exposure times. Validation of coculture screening data. (F) Flow cytometry analysis of cell death using Live/Dead Blue stain. FarRed CellTrace was used to label and distinguish fibroblasts. (G) Quantification of percent TNBC cell death in experiment described in panel (F). (H and I) Flow cytometry and quantitative analysis, as in panels (F and G) for Hs578T +/− WS1 and treated with etoposide. Error bars represent standard deviation of biological replicates. P ‐value calculated using t ‐test. Data are from three biological replicates and error bars. *** P ‐value

    Journal: Molecular Systems Biology

    Article Title: Tumor‐stroma interactions differentially alter drug sensitivity based on the origin of stromal cells

    doi: 10.15252/msb.20188322

    Figure Lengend Snippet: Coculture screen to identify tumor–stroma interactions reveals divergent interactions between fibroblasts and TNBC cells Schematic of screen design. TNBC cell lines were labeled with JC‐1, grown in monoculture or in coculture with primary fibroblast cells, and treated with one of 42 anticancer drugs. JC‐1 fluorescence was monitored using a fluorescence plate reader at 8‐hour intervals for 72 h. Representative images of BT‐20 cells (BL subtype) cocultured with HADF fibroblasts. BT‐20 cells labeled with JC‐1 dye; HADF labeled with a blue cell dye (CellTrace). Images taken before drug addition (B) or 96 h after exposure to 0.5 μM camptothecin (C). Kinetic trace of JC‐1 red fluorescence following exposure to camptothecin as in (B and C). Data are relative JC‐1 red fluorescence, normalized to the well's average prior to drug addition. Data represent mean ± standard deviation for five biological replicates. Total coculture screen data. Each blue dot represents a unique TNBC–fibroblast–drug measurement. 312,120 total measurements of drug response. Orange and purple dots highlight conditions validated in panels (F–I). Orange are HCC‐1143 (BL) cells cocultured with HCPF and treated with palbociclib. Purple dots are Hs578T (ML) cocultured with WS1 and exposed to etoposide. For colored dots, increasing size represents longer drug exposure times. Validation of coculture screening data. (F) Flow cytometry analysis of cell death using Live/Dead Blue stain. FarRed CellTrace was used to label and distinguish fibroblasts. (G) Quantification of percent TNBC cell death in experiment described in panel (F). (H and I) Flow cytometry and quantitative analysis, as in panels (F and G) for Hs578T +/− WS1 and treated with etoposide. Error bars represent standard deviation of biological replicates. P ‐value calculated using t ‐test. Data are from three biological replicates and error bars. *** P ‐value

    Article Snippet: 1,500 stained cells were plated in 40 μl FluoroBrite media (Thermofisher # A1896701), supplemented with 10% FBS, 2 mM glutamine, and penicillin/streptomycin, in a Greiner clear 384‐well plate (#781986) and allowed to adhere for 3 h. Cancer cell lines were then trypsinized and stained with 1.5 μg/ml (final concentration) JC‐1 (Thermofisher # T3168) in FluoroBrite at a concentration of 1 × 106 cell/ml for 20 min at 37°C.

    Techniques: Labeling, Fluorescence, Standard Deviation, Flow Cytometry, Cytometry, Staining

    Mitochondrial activity in subpopulations of hPSC. a Double label staining of live cells with TMRM and stem cell surface marker CD9. Cells at the edges of colonies stain most strongly with antibody and dye. Scale bar = 100 micron. b Flow cytometry analysis of TMRM staining in GCTM-2 high CD9 high EPCAM high (HHH), GCTM-2 low CD9 low (LOW), and unsorted (GEN) population. c Flow cytometry analysis of green (left panel) and red (right panel) JC-1 dye emission in GCTM-2 high CD9 high EPCAM high (HHH), GCTM-2 low CD9 low (LOW), and unsorted (GEN) population. GCTM-2 high CD9 high EPCAM high cells display highest ratio of green to red emission, indicative of high mitochondrial membrane potential. Results in a display representative outcomes from three experiments.

    Journal: Nature Communications

    Article Title: Unique properties of a subset of human pluripotent stem cells with high capacity for self-renewal

    doi: 10.1038/s41467-020-16214-8

    Figure Lengend Snippet: Mitochondrial activity in subpopulations of hPSC. a Double label staining of live cells with TMRM and stem cell surface marker CD9. Cells at the edges of colonies stain most strongly with antibody and dye. Scale bar = 100 micron. b Flow cytometry analysis of TMRM staining in GCTM-2 high CD9 high EPCAM high (HHH), GCTM-2 low CD9 low (LOW), and unsorted (GEN) population. c Flow cytometry analysis of green (left panel) and red (right panel) JC-1 dye emission in GCTM-2 high CD9 high EPCAM high (HHH), GCTM-2 low CD9 low (LOW), and unsorted (GEN) population. GCTM-2 high CD9 high EPCAM high cells display highest ratio of green to red emission, indicative of high mitochondrial membrane potential. Results in a display representative outcomes from three experiments.

    Article Snippet: 1 × 106 single cells that were stained with antibodies were resuspended in 1 mL of mTeSR1 supplemented with 250 nM TMRM (Thermo Fisher, Cat. #T668) or 0.3 μg/mL JC-1 (Thermo Fisher, Cat. #T3168) and incubated in the incubator for 30 min.

    Techniques: Activity Assay, Staining, Marker, Flow Cytometry

    Mitochondrial polarization does not shift with short-term hair cell stimulation. Mean JC-1 fluorescence ratio measured following 90 min of orbital stimulation. Larvae were incubated in JC-1 just prior to orbital shaking. Control: 0.58 ± 0.17; Orbital Shaker: 0.57 ± 0.08; mean ratio ± SD, n = 10 fish per group, 2–3 neuromasts per fish; Mann-Whitney U test, p = 0.91).

    Journal: eLife

    Article Title: Cumulative mitochondrial activity correlates with ototoxin susceptibility in zebrafish mechanosensory hair cells

    doi: 10.7554/eLife.38062

    Figure Lengend Snippet: Mitochondrial polarization does not shift with short-term hair cell stimulation. Mean JC-1 fluorescence ratio measured following 90 min of orbital stimulation. Larvae were incubated in JC-1 just prior to orbital shaking. Control: 0.58 ± 0.17; Orbital Shaker: 0.57 ± 0.08; mean ratio ± SD, n = 10 fish per group, 2–3 neuromasts per fish; Mann-Whitney U test, p = 0.91).

    Article Snippet: The same imaging and analysis protocol was followed for static imaging of zebrafish labeled with JC-1 dye (ThermoFisher).

    Techniques: Cell Stimulation, Fluorescence, Incubation, Fluorescence In Situ Hybridization, MANN-WHITNEY

    Acute mitochondrial activity is reduced in the absence of MET. ( A, B ) Maximum projections of hair cells from WT/Het and sputnik mutant siblings incubated in JC-1 dye. Hair cells were imaged from a dorsal view, as indicated in the schematic shown in Figure 1—figure supplement 1B . ( C ) Mean JC-1 fluorescence plotted as a ratio of red:green. WT/Het: 0.25 ± 0.24 n = 8 fish; Mutant: 0.05 ± 0.07 n = 8 fish; mean ratio ± SD. Mann-Whitney U test was used to assess significance. Value for each fish represents the mean of 3 neuromasts. Scale bar = 5 μm.

    Journal: eLife

    Article Title: Cumulative mitochondrial activity correlates with ototoxin susceptibility in zebrafish mechanosensory hair cells

    doi: 10.7554/eLife.38062

    Figure Lengend Snippet: Acute mitochondrial activity is reduced in the absence of MET. ( A, B ) Maximum projections of hair cells from WT/Het and sputnik mutant siblings incubated in JC-1 dye. Hair cells were imaged from a dorsal view, as indicated in the schematic shown in Figure 1—figure supplement 1B . ( C ) Mean JC-1 fluorescence plotted as a ratio of red:green. WT/Het: 0.25 ± 0.24 n = 8 fish; Mutant: 0.05 ± 0.07 n = 8 fish; mean ratio ± SD. Mann-Whitney U test was used to assess significance. Value for each fish represents the mean of 3 neuromasts. Scale bar = 5 μm.

    Article Snippet: The same imaging and analysis protocol was followed for static imaging of zebrafish labeled with JC-1 dye (ThermoFisher).

    Techniques: Activity Assay, Mutagenesis, Incubation, Fluorescence, Fluorescence In Situ Hybridization, MANN-WHITNEY

    TET enhanced the pro-apoptotic effect of PTX in SKOV3/PTX cells. SKOV3/PTX cells were treated with PTX (1 μM) or/and 2 μM TET for 72 h. ( A, B ) Apoptotic cells were detected by Annexin V/PI double staining and flow cytometry in SKOV3/PTX cells. ( C ) JC-1 staining was used to determine MMP loss. ( D ) Expression levels of Bax, Bcl-2 and cleaved caspase 3 in SKOV3/PTX cells were detected with Western blotting. ( E – G ) The relative expressions of Bax, Bcl-2 and cleaved caspase 3 were quantified via normalization to β-actin. *P

    Journal: OncoTargets and therapy

    Article Title: Tetrandrine Reverses Paclitaxel Resistance in Human Ovarian Cancer via Inducing Apoptosis, Cell Cycle Arrest Through β-Catenin Pathway

    doi: 10.2147/OTT.S235533

    Figure Lengend Snippet: TET enhanced the pro-apoptotic effect of PTX in SKOV3/PTX cells. SKOV3/PTX cells were treated with PTX (1 μM) or/and 2 μM TET for 72 h. ( A, B ) Apoptotic cells were detected by Annexin V/PI double staining and flow cytometry in SKOV3/PTX cells. ( C ) JC-1 staining was used to determine MMP loss. ( D ) Expression levels of Bax, Bcl-2 and cleaved caspase 3 in SKOV3/PTX cells were detected with Western blotting. ( E – G ) The relative expressions of Bax, Bcl-2 and cleaved caspase 3 were quantified via normalization to β-actin. *P

    Article Snippet: JC-1 Staining Mitochondrial membrane potential (MMP) was assessed by cationic dye JC-1 using the MMP assay kit (Thermo Fisher Scientific) according to the manufacturer’s protocol.

    Techniques: Double Staining, Flow Cytometry, Staining, Expressing, Western Blot