atp based celltiter glo luminescent cell viability assay solution  (Promega)

 
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    Promega atp based celltiter glo luminescent cell viability assay solution
    Doxorubicin (DOX) and etoposide (ETO) differentially regulate mitochondrial mass via respiration, cell adhesion, and cell death. ( A – C ) HK-2 cells were treated with DOX (1 μM) or ETO (50 μM) for 72 h. (A) Cell viability was measured using MTT assays, and signals were detected using a microplate reader. ( B ) Expression of PARP1 (116 kDa), cleaved-PARP1 (C-PARP1; 89 kDa and 55 kDa), cleaved-caspase 3 (19 kDa), and BCL2 (26 kDa) were determined using western blot analysis of whole cell extracts. β-Actin was used as a loading control. ( C ) HK-2 and treated cells were counted using a hemocytometer, and same number of cells seeded on plates. Caspase 3/7 activity was measured using <t>Caspase-Glo</t> 3/7 Assays, and signals were detected using a microplate reader, which measured luminescence intensity. ( D–J ) HK-2 cells were treated with DOX (1 μM) and ETO (50 μM) for 72 h, followed by treatment with CCCP (50 μM) for 5 min. ( D ) Mitochondrial mass and respiration were measured using MitoTracker Green FM and Red CMXRos double staining, respectively. Detection was facilitated by FACS analysis, and the percentage of fluorescence-positive cells was measured using dot plot analysis. The histogram shows fluorescence intensities. ( E ) Mitochondrial mass (monomer) and outer membrane potential (ΔΨm; aggregation) were measured using JC-1 staining and detection by FACS analysis. Fluorescence intensities are presented in the histogram. ( F ) HK-2 cells and treated cells were counted using a hemocytometer, and same number of cells was seeded on plates. Cytosolic <t>ATP</t> levels were measured using ATP-based <t>CellTiter-Glo</t> Luminescent Cell Viability Assays and were detected using a microplate reader. ( G ) HK-2 and treated cells were counted using a hemocytometer, and the same number of cells was seeded on a device that allows measurement of the cell-adhesion area by real-time monitoring of impedance, using an xCELLigence System, during the 2.5-h study period. ( H ) Analysis of DOX- and ETO-dependent cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double-staining. Necrotic and/or apoptotic cells were detected by FACS analysis. The histogram shows the percentage of cells, and represents the average of the results of three independent experiments (* P
    Atp Based Celltiter Glo Luminescent Cell Viability Assay Solution, supplied by Promega, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/atp based celltiter glo luminescent cell viability assay solution/product/Promega
    Average 79 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    atp based celltiter glo luminescent cell viability assay solution - by Bioz Stars, 2020-01
    79/100 stars

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    1) Product Images from "Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53"

    Article Title: Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53

    Journal: Scientific Reports

    doi: 10.1038/srep15798

    Doxorubicin (DOX) and etoposide (ETO) differentially regulate mitochondrial mass via respiration, cell adhesion, and cell death. ( A – C ) HK-2 cells were treated with DOX (1 μM) or ETO (50 μM) for 72 h. (A) Cell viability was measured using MTT assays, and signals were detected using a microplate reader. ( B ) Expression of PARP1 (116 kDa), cleaved-PARP1 (C-PARP1; 89 kDa and 55 kDa), cleaved-caspase 3 (19 kDa), and BCL2 (26 kDa) were determined using western blot analysis of whole cell extracts. β-Actin was used as a loading control. ( C ) HK-2 and treated cells were counted using a hemocytometer, and same number of cells seeded on plates. Caspase 3/7 activity was measured using Caspase-Glo 3/7 Assays, and signals were detected using a microplate reader, which measured luminescence intensity. ( D–J ) HK-2 cells were treated with DOX (1 μM) and ETO (50 μM) for 72 h, followed by treatment with CCCP (50 μM) for 5 min. ( D ) Mitochondrial mass and respiration were measured using MitoTracker Green FM and Red CMXRos double staining, respectively. Detection was facilitated by FACS analysis, and the percentage of fluorescence-positive cells was measured using dot plot analysis. The histogram shows fluorescence intensities. ( E ) Mitochondrial mass (monomer) and outer membrane potential (ΔΨm; aggregation) were measured using JC-1 staining and detection by FACS analysis. Fluorescence intensities are presented in the histogram. ( F ) HK-2 cells and treated cells were counted using a hemocytometer, and same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays and were detected using a microplate reader. ( G ) HK-2 and treated cells were counted using a hemocytometer, and the same number of cells was seeded on a device that allows measurement of the cell-adhesion area by real-time monitoring of impedance, using an xCELLigence System, during the 2.5-h study period. ( H ) Analysis of DOX- and ETO-dependent cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double-staining. Necrotic and/or apoptotic cells were detected by FACS analysis. The histogram shows the percentage of cells, and represents the average of the results of three independent experiments (* P
    Figure Legend Snippet: Doxorubicin (DOX) and etoposide (ETO) differentially regulate mitochondrial mass via respiration, cell adhesion, and cell death. ( A – C ) HK-2 cells were treated with DOX (1 μM) or ETO (50 μM) for 72 h. (A) Cell viability was measured using MTT assays, and signals were detected using a microplate reader. ( B ) Expression of PARP1 (116 kDa), cleaved-PARP1 (C-PARP1; 89 kDa and 55 kDa), cleaved-caspase 3 (19 kDa), and BCL2 (26 kDa) were determined using western blot analysis of whole cell extracts. β-Actin was used as a loading control. ( C ) HK-2 and treated cells were counted using a hemocytometer, and same number of cells seeded on plates. Caspase 3/7 activity was measured using Caspase-Glo 3/7 Assays, and signals were detected using a microplate reader, which measured luminescence intensity. ( D–J ) HK-2 cells were treated with DOX (1 μM) and ETO (50 μM) for 72 h, followed by treatment with CCCP (50 μM) for 5 min. ( D ) Mitochondrial mass and respiration were measured using MitoTracker Green FM and Red CMXRos double staining, respectively. Detection was facilitated by FACS analysis, and the percentage of fluorescence-positive cells was measured using dot plot analysis. The histogram shows fluorescence intensities. ( E ) Mitochondrial mass (monomer) and outer membrane potential (ΔΨm; aggregation) were measured using JC-1 staining and detection by FACS analysis. Fluorescence intensities are presented in the histogram. ( F ) HK-2 cells and treated cells were counted using a hemocytometer, and same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays and were detected using a microplate reader. ( G ) HK-2 and treated cells were counted using a hemocytometer, and the same number of cells was seeded on a device that allows measurement of the cell-adhesion area by real-time monitoring of impedance, using an xCELLigence System, during the 2.5-h study period. ( H ) Analysis of DOX- and ETO-dependent cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double-staining. Necrotic and/or apoptotic cells were detected by FACS analysis. The histogram shows the percentage of cells, and represents the average of the results of three independent experiments (* P

    Techniques Used: MTT Assay, Expressing, Western Blot, Activity Assay, Double Staining, FACS, Fluorescence, Staining

    Treatment with a ROS scavenger suppresses doxorubicin (DOX)-induced cytosolic adenosine triphosphate (cATP) production, DNA damage, mitochondrial hyper-activation, and necrosis. HK-2 cells were treated with a ROS scavenger (N-acetyl cysteine, NAC, 5 mM), DOX (1 μM), or both DOX and NAC for 72 h. ( A , B ) Cytosolic ROS and mitochondrial ROS levels were measured using DCF-DA and MitoSOX, staining, respectively, and signals were detected by FACS analysis. ( C ) Expression levels of PARP1 (116 kDa), C-PARP1 (89 kDa), γ-H2AX ser139 (15 kDa), and H2AX were measured by western blot analysis in whole cell extracts. β-Actin was used as a loading control. The gels were electrophoresed under the same experimental conditions. ( D , E ) Mitochondrial respiration and cATP production were measured using MitoTracker Red CMXRos staining and ATP-based CellTiter-Glo Luminescent Cell Viability Assays, and signals were detected using FACS analysis or analysis on a microplate reader, respectively. ( F ) Morphological changes were measured using phase-contrast microscopy (original magnification, 100×). ( G ) Analysis of cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double staining. Necrotic and/or apoptotic cells were detected using FACS analysis. The histogram shows the percentage of cells in each group, as the average of the results of three independent experiments (* P
    Figure Legend Snippet: Treatment with a ROS scavenger suppresses doxorubicin (DOX)-induced cytosolic adenosine triphosphate (cATP) production, DNA damage, mitochondrial hyper-activation, and necrosis. HK-2 cells were treated with a ROS scavenger (N-acetyl cysteine, NAC, 5 mM), DOX (1 μM), or both DOX and NAC for 72 h. ( A , B ) Cytosolic ROS and mitochondrial ROS levels were measured using DCF-DA and MitoSOX, staining, respectively, and signals were detected by FACS analysis. ( C ) Expression levels of PARP1 (116 kDa), C-PARP1 (89 kDa), γ-H2AX ser139 (15 kDa), and H2AX were measured by western blot analysis in whole cell extracts. β-Actin was used as a loading control. The gels were electrophoresed under the same experimental conditions. ( D , E ) Mitochondrial respiration and cATP production were measured using MitoTracker Red CMXRos staining and ATP-based CellTiter-Glo Luminescent Cell Viability Assays, and signals were detected using FACS analysis or analysis on a microplate reader, respectively. ( F ) Morphological changes were measured using phase-contrast microscopy (original magnification, 100×). ( G ) Analysis of cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double staining. Necrotic and/or apoptotic cells were detected using FACS analysis. The histogram shows the percentage of cells in each group, as the average of the results of three independent experiments (* P

    Techniques Used: Activation Assay, Staining, FACS, Expressing, Western Blot, Microscopy, Double Staining

    Inhibition of PARP1 suppresses doxorubicin (DOX)-induced DNA damage, mitochondrial hyper-activation, and necrosis. ( A–J ) HK-2 cells were treated with a pharmacological PARP1 inhibitor (PJ-34, 10 μM), DOX (1 μM), or both PJ-34 and DOX for 72 h. (A) Expressions of PARP1 (116 kDa), C-PARP1 (89 kDa), γ-H2AX ser139 (15 kDa), H2AX (15 kDa), 53BP1 (350 kDa), p-ATM ser1981 (350 kDa), ATM (350 kDa), p-CHK2 Thr68 (62 kDa), and CHK2 (62 kDa) proteins were measured by western blot in whole cell extracts. ( B ) Levels of p-ATM ser1981 and γ-H2AX ser139 in the nucleus were measured using immunofluorescence staining and Cellomics ArrayScan HCS Reader. ( C ) Analysis of cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double staining. Necrotic and/or apoptotic cells were detected by FACS. ( D ) Mitochondrial respiration levels were measured using MitoTracker Red CMXRos and signals were detected using FACS. ( E ) Genomic DNA was analyzed for mtDNA copy numbers using ND1- , ND4- , and GAPDH- specific primers and real-time PCR. ND1 and ND4 copy numbers were normalized to GAPDH copy numbers. ( F ) The same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays. ( G , H ) Cytosolic ROS and mitochondrial ROS levels were measured using DCF-DA and MitoSOX, respectively, and signals were detected by FACS. (I and J) Cytosolic NO and secreted NO levels were measured using DAF-FM and an NO detection kit. ( K , N ) HK-2 cells were transfected with SC-siRNA (5 nM) or PARP1 -siRNA (5 nM) and treated with DOX for 72 h. ( K ) The same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assay. ( L ) Cytosolic ROS levels were measured using DCF-DA and were detected using FACS. ( M ) Cell viability was measured using MTT assay, and signals were detected by a microplate reader. ( N ) Necrotic cell death was measured using PI staining, and signals were detected by FACS. (* P
    Figure Legend Snippet: Inhibition of PARP1 suppresses doxorubicin (DOX)-induced DNA damage, mitochondrial hyper-activation, and necrosis. ( A–J ) HK-2 cells were treated with a pharmacological PARP1 inhibitor (PJ-34, 10 μM), DOX (1 μM), or both PJ-34 and DOX for 72 h. (A) Expressions of PARP1 (116 kDa), C-PARP1 (89 kDa), γ-H2AX ser139 (15 kDa), H2AX (15 kDa), 53BP1 (350 kDa), p-ATM ser1981 (350 kDa), ATM (350 kDa), p-CHK2 Thr68 (62 kDa), and CHK2 (62 kDa) proteins were measured by western blot in whole cell extracts. ( B ) Levels of p-ATM ser1981 and γ-H2AX ser139 in the nucleus were measured using immunofluorescence staining and Cellomics ArrayScan HCS Reader. ( C ) Analysis of cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double staining. Necrotic and/or apoptotic cells were detected by FACS. ( D ) Mitochondrial respiration levels were measured using MitoTracker Red CMXRos and signals were detected using FACS. ( E ) Genomic DNA was analyzed for mtDNA copy numbers using ND1- , ND4- , and GAPDH- specific primers and real-time PCR. ND1 and ND4 copy numbers were normalized to GAPDH copy numbers. ( F ) The same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays. ( G , H ) Cytosolic ROS and mitochondrial ROS levels were measured using DCF-DA and MitoSOX, respectively, and signals were detected by FACS. (I and J) Cytosolic NO and secreted NO levels were measured using DAF-FM and an NO detection kit. ( K , N ) HK-2 cells were transfected with SC-siRNA (5 nM) or PARP1 -siRNA (5 nM) and treated with DOX for 72 h. ( K ) The same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assay. ( L ) Cytosolic ROS levels were measured using DCF-DA and were detected using FACS. ( M ) Cell viability was measured using MTT assay, and signals were detected by a microplate reader. ( N ) Necrotic cell death was measured using PI staining, and signals were detected by FACS. (* P

    Techniques Used: Inhibition, Activation Assay, Western Blot, Immunofluorescence, Staining, Double Staining, FACS, Real-time Polymerase Chain Reaction, Transfection, Cell Viability Assay, MTT Assay

    Adenosine triphosphate (ATP) treatment induces DNA damage and necrosis at the early stage and apoptosis at the late stage, but does not alter cytosolic reactive oxygen species (cROS) and mitochondrial reactive oxygen species (mROS) levels. HK-2 cells were treated with ATP (1 mM) for 24, 48, and 72 h. ( A ) Cell viability was measured using MTT assays. HK-2 and ATP-treated cells were counted using a hemocytometer and the same number of cells was seeded on plates. Cytosolic ATP was measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays. Signals were detected using a microplate reader. ( B ) Expression levels of PARP1 (116 kDa), C-PARP1 (89 kDa and 55 kDa), γ-H2AX ser139 (15 kDa), and H2AX (15 kDa) proteins were measured by western blot analysis in whole cell extracts. β-Actin was used as a loading control. ( C ) HK-2 and ATP-treated cells were counted using a hemocytometer and the same number of cells was seeded on plates. Caspase 3/7 activity was measured by Caspase-Glo 3/7 Assays, and signals were detected using a microplate reader, which measured luminescence intensity. Representative images are shown. ( D , E ) cROS, mROS, and cytosolic NO levels were measured using DCF-DA, MitoSOX, and DAF-FM staining, respectively, and signals were detected by FACS analysis. ( F ) Cell death, including necrosis (PI-positive cells) and apoptosis (Annexin V-positive cells), was measured using Annexin V or PI staining. Signals were detected by FACS analysis. The histogram shows the percentage of PI- and Annexin V-positive cells, as the average of the results of three independent experiments (*P
    Figure Legend Snippet: Adenosine triphosphate (ATP) treatment induces DNA damage and necrosis at the early stage and apoptosis at the late stage, but does not alter cytosolic reactive oxygen species (cROS) and mitochondrial reactive oxygen species (mROS) levels. HK-2 cells were treated with ATP (1 mM) for 24, 48, and 72 h. ( A ) Cell viability was measured using MTT assays. HK-2 and ATP-treated cells were counted using a hemocytometer and the same number of cells was seeded on plates. Cytosolic ATP was measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays. Signals were detected using a microplate reader. ( B ) Expression levels of PARP1 (116 kDa), C-PARP1 (89 kDa and 55 kDa), γ-H2AX ser139 (15 kDa), and H2AX (15 kDa) proteins were measured by western blot analysis in whole cell extracts. β-Actin was used as a loading control. ( C ) HK-2 and ATP-treated cells were counted using a hemocytometer and the same number of cells was seeded on plates. Caspase 3/7 activity was measured by Caspase-Glo 3/7 Assays, and signals were detected using a microplate reader, which measured luminescence intensity. Representative images are shown. ( D , E ) cROS, mROS, and cytosolic NO levels were measured using DCF-DA, MitoSOX, and DAF-FM staining, respectively, and signals were detected by FACS analysis. ( F ) Cell death, including necrosis (PI-positive cells) and apoptosis (Annexin V-positive cells), was measured using Annexin V or PI staining. Signals were detected by FACS analysis. The histogram shows the percentage of PI- and Annexin V-positive cells, as the average of the results of three independent experiments (*P

    Techniques Used: MTT Assay, Expressing, Western Blot, Activity Assay, Staining, FACS

    Related Articles

    Cell Viability Assay:

    Article Title: Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53
    Article Snippet: Harvested cells were counted using a hemocytometer (Paul Marienfeld GmbH & Co., KG, Bad Mergentheim, Germany) and seeded at a density of 1 × 104 cells per well in 96-well plates (Greiner Bio-One, Frickenhausen, Germany). .. Next, 50 μL ATP-based CellTiter-Glo Luminescent Cell Viability Assay solution (Promega, Madison, WI, USA) was added, and cells were incubated for 10 min. .. The luminescence intensity was measured using a Molecular Devices SPECTRAmax GEMINI fluorescence microplate reader (Molecular Devices Inc.) and/or a Fuji LAS-3000 system (Fujifilm, Tokyo, Japan).

    Centrifugation:

    Article Title: Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53
    Article Snippet: Cells were harvested by centrifugation at 200 × g for 3 min to remove extracellular ATP. .. Next, 50 μL ATP-based CellTiter-Glo Luminescent Cell Viability Assay solution (Promega, Madison, WI, USA) was added, and cells were incubated for 10 min.

    Incubation:

    Article Title: Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53
    Article Snippet: Harvested cells were counted using a hemocytometer (Paul Marienfeld GmbH & Co., KG, Bad Mergentheim, Germany) and seeded at a density of 1 × 104 cells per well in 96-well plates (Greiner Bio-One, Frickenhausen, Germany). .. Next, 50 μL ATP-based CellTiter-Glo Luminescent Cell Viability Assay solution (Promega, Madison, WI, USA) was added, and cells were incubated for 10 min. .. The luminescence intensity was measured using a Molecular Devices SPECTRAmax GEMINI fluorescence microplate reader (Molecular Devices Inc.) and/or a Fuji LAS-3000 system (Fujifilm, Tokyo, Japan).

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    Promega atp based celltiter glo luminescent cell viability assay solution
    Doxorubicin (DOX) and etoposide (ETO) differentially regulate mitochondrial mass via respiration, cell adhesion, and cell death. ( A – C ) HK-2 cells were treated with DOX (1 μM) or ETO (50 μM) for 72 h. (A) Cell viability was measured using MTT assays, and signals were detected using a microplate reader. ( B ) Expression of PARP1 (116 kDa), cleaved-PARP1 (C-PARP1; 89 kDa and 55 kDa), cleaved-caspase 3 (19 kDa), and BCL2 (26 kDa) were determined using western blot analysis of whole cell extracts. β-Actin was used as a loading control. ( C ) HK-2 and treated cells were counted using a hemocytometer, and same number of cells seeded on plates. Caspase 3/7 activity was measured using <t>Caspase-Glo</t> 3/7 Assays, and signals were detected using a microplate reader, which measured luminescence intensity. ( D–J ) HK-2 cells were treated with DOX (1 μM) and ETO (50 μM) for 72 h, followed by treatment with CCCP (50 μM) for 5 min. ( D ) Mitochondrial mass and respiration were measured using MitoTracker Green FM and Red CMXRos double staining, respectively. Detection was facilitated by FACS analysis, and the percentage of fluorescence-positive cells was measured using dot plot analysis. The histogram shows fluorescence intensities. ( E ) Mitochondrial mass (monomer) and outer membrane potential (ΔΨm; aggregation) were measured using JC-1 staining and detection by FACS analysis. Fluorescence intensities are presented in the histogram. ( F ) HK-2 cells and treated cells were counted using a hemocytometer, and same number of cells was seeded on plates. Cytosolic <t>ATP</t> levels were measured using ATP-based <t>CellTiter-Glo</t> Luminescent Cell Viability Assays and were detected using a microplate reader. ( G ) HK-2 and treated cells were counted using a hemocytometer, and the same number of cells was seeded on a device that allows measurement of the cell-adhesion area by real-time monitoring of impedance, using an xCELLigence System, during the 2.5-h study period. ( H ) Analysis of DOX- and ETO-dependent cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double-staining. Necrotic and/or apoptotic cells were detected by FACS analysis. The histogram shows the percentage of cells, and represents the average of the results of three independent experiments (* P
    Atp Based Celltiter Glo Luminescent Cell Viability Assay Solution, supplied by Promega, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/atp based celltiter glo luminescent cell viability assay solution/product/Promega
    Average 79 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    atp based celltiter glo luminescent cell viability assay solution - by Bioz Stars, 2020-01
    79/100 stars
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    Doxorubicin (DOX) and etoposide (ETO) differentially regulate mitochondrial mass via respiration, cell adhesion, and cell death. ( A – C ) HK-2 cells were treated with DOX (1 μM) or ETO (50 μM) for 72 h. (A) Cell viability was measured using MTT assays, and signals were detected using a microplate reader. ( B ) Expression of PARP1 (116 kDa), cleaved-PARP1 (C-PARP1; 89 kDa and 55 kDa), cleaved-caspase 3 (19 kDa), and BCL2 (26 kDa) were determined using western blot analysis of whole cell extracts. β-Actin was used as a loading control. ( C ) HK-2 and treated cells were counted using a hemocytometer, and same number of cells seeded on plates. Caspase 3/7 activity was measured using Caspase-Glo 3/7 Assays, and signals were detected using a microplate reader, which measured luminescence intensity. ( D–J ) HK-2 cells were treated with DOX (1 μM) and ETO (50 μM) for 72 h, followed by treatment with CCCP (50 μM) for 5 min. ( D ) Mitochondrial mass and respiration were measured using MitoTracker Green FM and Red CMXRos double staining, respectively. Detection was facilitated by FACS analysis, and the percentage of fluorescence-positive cells was measured using dot plot analysis. The histogram shows fluorescence intensities. ( E ) Mitochondrial mass (monomer) and outer membrane potential (ΔΨm; aggregation) were measured using JC-1 staining and detection by FACS analysis. Fluorescence intensities are presented in the histogram. ( F ) HK-2 cells and treated cells were counted using a hemocytometer, and same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays and were detected using a microplate reader. ( G ) HK-2 and treated cells were counted using a hemocytometer, and the same number of cells was seeded on a device that allows measurement of the cell-adhesion area by real-time monitoring of impedance, using an xCELLigence System, during the 2.5-h study period. ( H ) Analysis of DOX- and ETO-dependent cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double-staining. Necrotic and/or apoptotic cells were detected by FACS analysis. The histogram shows the percentage of cells, and represents the average of the results of three independent experiments (* P

    Journal: Scientific Reports

    Article Title: Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53

    doi: 10.1038/srep15798

    Figure Lengend Snippet: Doxorubicin (DOX) and etoposide (ETO) differentially regulate mitochondrial mass via respiration, cell adhesion, and cell death. ( A – C ) HK-2 cells were treated with DOX (1 μM) or ETO (50 μM) for 72 h. (A) Cell viability was measured using MTT assays, and signals were detected using a microplate reader. ( B ) Expression of PARP1 (116 kDa), cleaved-PARP1 (C-PARP1; 89 kDa and 55 kDa), cleaved-caspase 3 (19 kDa), and BCL2 (26 kDa) were determined using western blot analysis of whole cell extracts. β-Actin was used as a loading control. ( C ) HK-2 and treated cells were counted using a hemocytometer, and same number of cells seeded on plates. Caspase 3/7 activity was measured using Caspase-Glo 3/7 Assays, and signals were detected using a microplate reader, which measured luminescence intensity. ( D–J ) HK-2 cells were treated with DOX (1 μM) and ETO (50 μM) for 72 h, followed by treatment with CCCP (50 μM) for 5 min. ( D ) Mitochondrial mass and respiration were measured using MitoTracker Green FM and Red CMXRos double staining, respectively. Detection was facilitated by FACS analysis, and the percentage of fluorescence-positive cells was measured using dot plot analysis. The histogram shows fluorescence intensities. ( E ) Mitochondrial mass (monomer) and outer membrane potential (ΔΨm; aggregation) were measured using JC-1 staining and detection by FACS analysis. Fluorescence intensities are presented in the histogram. ( F ) HK-2 cells and treated cells were counted using a hemocytometer, and same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays and were detected using a microplate reader. ( G ) HK-2 and treated cells were counted using a hemocytometer, and the same number of cells was seeded on a device that allows measurement of the cell-adhesion area by real-time monitoring of impedance, using an xCELLigence System, during the 2.5-h study period. ( H ) Analysis of DOX- and ETO-dependent cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double-staining. Necrotic and/or apoptotic cells were detected by FACS analysis. The histogram shows the percentage of cells, and represents the average of the results of three independent experiments (* P

    Article Snippet: Next, 50 μL ATP-based CellTiter-Glo Luminescent Cell Viability Assay solution (Promega, Madison, WI, USA) was added, and cells were incubated for 10 min.

    Techniques: MTT Assay, Expressing, Western Blot, Activity Assay, Double Staining, FACS, Fluorescence, Staining

    Treatment with a ROS scavenger suppresses doxorubicin (DOX)-induced cytosolic adenosine triphosphate (cATP) production, DNA damage, mitochondrial hyper-activation, and necrosis. HK-2 cells were treated with a ROS scavenger (N-acetyl cysteine, NAC, 5 mM), DOX (1 μM), or both DOX and NAC for 72 h. ( A , B ) Cytosolic ROS and mitochondrial ROS levels were measured using DCF-DA and MitoSOX, staining, respectively, and signals were detected by FACS analysis. ( C ) Expression levels of PARP1 (116 kDa), C-PARP1 (89 kDa), γ-H2AX ser139 (15 kDa), and H2AX were measured by western blot analysis in whole cell extracts. β-Actin was used as a loading control. The gels were electrophoresed under the same experimental conditions. ( D , E ) Mitochondrial respiration and cATP production were measured using MitoTracker Red CMXRos staining and ATP-based CellTiter-Glo Luminescent Cell Viability Assays, and signals were detected using FACS analysis or analysis on a microplate reader, respectively. ( F ) Morphological changes were measured using phase-contrast microscopy (original magnification, 100×). ( G ) Analysis of cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double staining. Necrotic and/or apoptotic cells were detected using FACS analysis. The histogram shows the percentage of cells in each group, as the average of the results of three independent experiments (* P

    Journal: Scientific Reports

    Article Title: Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53

    doi: 10.1038/srep15798

    Figure Lengend Snippet: Treatment with a ROS scavenger suppresses doxorubicin (DOX)-induced cytosolic adenosine triphosphate (cATP) production, DNA damage, mitochondrial hyper-activation, and necrosis. HK-2 cells were treated with a ROS scavenger (N-acetyl cysteine, NAC, 5 mM), DOX (1 μM), or both DOX and NAC for 72 h. ( A , B ) Cytosolic ROS and mitochondrial ROS levels were measured using DCF-DA and MitoSOX, staining, respectively, and signals were detected by FACS analysis. ( C ) Expression levels of PARP1 (116 kDa), C-PARP1 (89 kDa), γ-H2AX ser139 (15 kDa), and H2AX were measured by western blot analysis in whole cell extracts. β-Actin was used as a loading control. The gels were electrophoresed under the same experimental conditions. ( D , E ) Mitochondrial respiration and cATP production were measured using MitoTracker Red CMXRos staining and ATP-based CellTiter-Glo Luminescent Cell Viability Assays, and signals were detected using FACS analysis or analysis on a microplate reader, respectively. ( F ) Morphological changes were measured using phase-contrast microscopy (original magnification, 100×). ( G ) Analysis of cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double staining. Necrotic and/or apoptotic cells were detected using FACS analysis. The histogram shows the percentage of cells in each group, as the average of the results of three independent experiments (* P

    Article Snippet: Next, 50 μL ATP-based CellTiter-Glo Luminescent Cell Viability Assay solution (Promega, Madison, WI, USA) was added, and cells were incubated for 10 min.

    Techniques: Activation Assay, Staining, FACS, Expressing, Western Blot, Microscopy, Double Staining

    Inhibition of PARP1 suppresses doxorubicin (DOX)-induced DNA damage, mitochondrial hyper-activation, and necrosis. ( A–J ) HK-2 cells were treated with a pharmacological PARP1 inhibitor (PJ-34, 10 μM), DOX (1 μM), or both PJ-34 and DOX for 72 h. (A) Expressions of PARP1 (116 kDa), C-PARP1 (89 kDa), γ-H2AX ser139 (15 kDa), H2AX (15 kDa), 53BP1 (350 kDa), p-ATM ser1981 (350 kDa), ATM (350 kDa), p-CHK2 Thr68 (62 kDa), and CHK2 (62 kDa) proteins were measured by western blot in whole cell extracts. ( B ) Levels of p-ATM ser1981 and γ-H2AX ser139 in the nucleus were measured using immunofluorescence staining and Cellomics ArrayScan HCS Reader. ( C ) Analysis of cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double staining. Necrotic and/or apoptotic cells were detected by FACS. ( D ) Mitochondrial respiration levels were measured using MitoTracker Red CMXRos and signals were detected using FACS. ( E ) Genomic DNA was analyzed for mtDNA copy numbers using ND1- , ND4- , and GAPDH- specific primers and real-time PCR. ND1 and ND4 copy numbers were normalized to GAPDH copy numbers. ( F ) The same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays. ( G , H ) Cytosolic ROS and mitochondrial ROS levels were measured using DCF-DA and MitoSOX, respectively, and signals were detected by FACS. (I and J) Cytosolic NO and secreted NO levels were measured using DAF-FM and an NO detection kit. ( K , N ) HK-2 cells were transfected with SC-siRNA (5 nM) or PARP1 -siRNA (5 nM) and treated with DOX for 72 h. ( K ) The same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assay. ( L ) Cytosolic ROS levels were measured using DCF-DA and were detected using FACS. ( M ) Cell viability was measured using MTT assay, and signals were detected by a microplate reader. ( N ) Necrotic cell death was measured using PI staining, and signals were detected by FACS. (* P

    Journal: Scientific Reports

    Article Title: Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53

    doi: 10.1038/srep15798

    Figure Lengend Snippet: Inhibition of PARP1 suppresses doxorubicin (DOX)-induced DNA damage, mitochondrial hyper-activation, and necrosis. ( A–J ) HK-2 cells were treated with a pharmacological PARP1 inhibitor (PJ-34, 10 μM), DOX (1 μM), or both PJ-34 and DOX for 72 h. (A) Expressions of PARP1 (116 kDa), C-PARP1 (89 kDa), γ-H2AX ser139 (15 kDa), H2AX (15 kDa), 53BP1 (350 kDa), p-ATM ser1981 (350 kDa), ATM (350 kDa), p-CHK2 Thr68 (62 kDa), and CHK2 (62 kDa) proteins were measured by western blot in whole cell extracts. ( B ) Levels of p-ATM ser1981 and γ-H2AX ser139 in the nucleus were measured using immunofluorescence staining and Cellomics ArrayScan HCS Reader. ( C ) Analysis of cell death, including necrosis (Q1), apoptosis (Q4), and necrosis with apoptosis (Q2), as measured using Annexin V and PI double staining. Necrotic and/or apoptotic cells were detected by FACS. ( D ) Mitochondrial respiration levels were measured using MitoTracker Red CMXRos and signals were detected using FACS. ( E ) Genomic DNA was analyzed for mtDNA copy numbers using ND1- , ND4- , and GAPDH- specific primers and real-time PCR. ND1 and ND4 copy numbers were normalized to GAPDH copy numbers. ( F ) The same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays. ( G , H ) Cytosolic ROS and mitochondrial ROS levels were measured using DCF-DA and MitoSOX, respectively, and signals were detected by FACS. (I and J) Cytosolic NO and secreted NO levels were measured using DAF-FM and an NO detection kit. ( K , N ) HK-2 cells were transfected with SC-siRNA (5 nM) or PARP1 -siRNA (5 nM) and treated with DOX for 72 h. ( K ) The same number of cells was seeded on plates. Cytosolic ATP levels were measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assay. ( L ) Cytosolic ROS levels were measured using DCF-DA and were detected using FACS. ( M ) Cell viability was measured using MTT assay, and signals were detected by a microplate reader. ( N ) Necrotic cell death was measured using PI staining, and signals were detected by FACS. (* P

    Article Snippet: Next, 50 μL ATP-based CellTiter-Glo Luminescent Cell Viability Assay solution (Promega, Madison, WI, USA) was added, and cells were incubated for 10 min.

    Techniques: Inhibition, Activation Assay, Western Blot, Immunofluorescence, Staining, Double Staining, FACS, Real-time Polymerase Chain Reaction, Transfection, Cell Viability Assay, MTT Assay

    Adenosine triphosphate (ATP) treatment induces DNA damage and necrosis at the early stage and apoptosis at the late stage, but does not alter cytosolic reactive oxygen species (cROS) and mitochondrial reactive oxygen species (mROS) levels. HK-2 cells were treated with ATP (1 mM) for 24, 48, and 72 h. ( A ) Cell viability was measured using MTT assays. HK-2 and ATP-treated cells were counted using a hemocytometer and the same number of cells was seeded on plates. Cytosolic ATP was measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays. Signals were detected using a microplate reader. ( B ) Expression levels of PARP1 (116 kDa), C-PARP1 (89 kDa and 55 kDa), γ-H2AX ser139 (15 kDa), and H2AX (15 kDa) proteins were measured by western blot analysis in whole cell extracts. β-Actin was used as a loading control. ( C ) HK-2 and ATP-treated cells were counted using a hemocytometer and the same number of cells was seeded on plates. Caspase 3/7 activity was measured by Caspase-Glo 3/7 Assays, and signals were detected using a microplate reader, which measured luminescence intensity. Representative images are shown. ( D , E ) cROS, mROS, and cytosolic NO levels were measured using DCF-DA, MitoSOX, and DAF-FM staining, respectively, and signals were detected by FACS analysis. ( F ) Cell death, including necrosis (PI-positive cells) and apoptosis (Annexin V-positive cells), was measured using Annexin V or PI staining. Signals were detected by FACS analysis. The histogram shows the percentage of PI- and Annexin V-positive cells, as the average of the results of three independent experiments (*P

    Journal: Scientific Reports

    Article Title: Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53

    doi: 10.1038/srep15798

    Figure Lengend Snippet: Adenosine triphosphate (ATP) treatment induces DNA damage and necrosis at the early stage and apoptosis at the late stage, but does not alter cytosolic reactive oxygen species (cROS) and mitochondrial reactive oxygen species (mROS) levels. HK-2 cells were treated with ATP (1 mM) for 24, 48, and 72 h. ( A ) Cell viability was measured using MTT assays. HK-2 and ATP-treated cells were counted using a hemocytometer and the same number of cells was seeded on plates. Cytosolic ATP was measured using ATP-based CellTiter-Glo Luminescent Cell Viability Assays. Signals were detected using a microplate reader. ( B ) Expression levels of PARP1 (116 kDa), C-PARP1 (89 kDa and 55 kDa), γ-H2AX ser139 (15 kDa), and H2AX (15 kDa) proteins were measured by western blot analysis in whole cell extracts. β-Actin was used as a loading control. ( C ) HK-2 and ATP-treated cells were counted using a hemocytometer and the same number of cells was seeded on plates. Caspase 3/7 activity was measured by Caspase-Glo 3/7 Assays, and signals were detected using a microplate reader, which measured luminescence intensity. Representative images are shown. ( D , E ) cROS, mROS, and cytosolic NO levels were measured using DCF-DA, MitoSOX, and DAF-FM staining, respectively, and signals were detected by FACS analysis. ( F ) Cell death, including necrosis (PI-positive cells) and apoptosis (Annexin V-positive cells), was measured using Annexin V or PI staining. Signals were detected by FACS analysis. The histogram shows the percentage of PI- and Annexin V-positive cells, as the average of the results of three independent experiments (*P

    Article Snippet: Next, 50 μL ATP-based CellTiter-Glo Luminescent Cell Viability Assay solution (Promega, Madison, WI, USA) was added, and cells were incubated for 10 min.

    Techniques: MTT Assay, Expressing, Western Blot, Activity Assay, Staining, FACS