Imaging:Article Title: Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy
Article Snippet: .. Antimycin A (Sigma, cat. no. A8674); see REAGENT SETUP DOX (Sigma, cat. no. 44583); see REAGENT SETUP DETANONOate (cat. no. 82120); see REAGENT SETUP SOD-PEG (cat. no. S9549); see REAGENT SETUP MPMVECs RPMI (Invitrogen) medium supplemented with 10% FBS, essential amino acids, endothelial cell growth supplement and antibiotics DMEM (Invitrogen) supplemented with 10% FBS, nonessential amino acids, endothelial cell growth supplement and antibiotics Extracellular medium (ECM); see REAGENT SETUP Endothelial cell growth supplement (Upstate Biotech) Experimental imaging solution (ECM containing 0.25% BSA) containing sulfinpyrazone (Sigma) Annexin binding buffer (10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl 2 , pH 7.4) pEYFP-Mito (Clontech, BD Biosciences, cat. no. 6115-1) Basic Nucleofector kit for primary mammalian endothelial cells (Amaxa Biosystems, cat. no. VPI-1001) .. Antimycin A (Sigma, cat. no. A8674); see REAGENT SETUP DOX (Sigma, cat. no. 44583); see REAGENT SETUP DETANONOate (cat. no. 82120); see REAGENT SETUP SOD-PEG (cat. no. S9549); see REAGENT SETUP MPMVECs RPMI (Invitrogen) medium supplemented with 10% FBS, essential amino acids, endothelial cell growth supplement and antibiotics DMEM (Invitrogen) supplemented with 10% FBS, nonessential amino acids, endothelial cell growth supplement and antibiotics Extracellular medium (ECM); see REAGENT SETUP Endothelial cell growth supplement (Upstate Biotech) Experimental imaging solution (ECM containing 0.25% BSA) containing sulfinpyrazone (Sigma) Annexin binding buffer (10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl 2 , pH 7.4) pEYFP-Mito (Clontech, BD Biosciences, cat. no. 6115-1) Basic Nucleofector kit for primary mammalian endothelial cells (Amaxa Biosystems, cat. no. VPI-1001)
Article Title: Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy
Article Snippet: HCAECs growth medium (Cell Applications Inc., 212K-500) or an appropriate medium for growing cells of interest MitoSOX Red (Molecular Probes, Invitrogen, cat. no. {"type":"entrez-nucleotide","attrs":{"text":"M36008","term_id":"214108","term_text":"M36008"}} M36008 ) supplied in 50 μg vials; see REAGENT SETUP Sytox Green nucleic acid stain (Molecular Probes, Invitrogen, cat. no. S7020); see REAGENT SETUP Allophycocyanin (APC)-Annexin V (cat. no. {"type":"entrez-nucleotide","attrs":{"text":"A35110","term_id":"1926769","term_text":"A35110"}} A35110 ) and Alexa Fluor 647-Annexin V (cat. no. A23204; Molecular Probes, Invitrogen) (use directly from supplied stock). .. Antimycin A (Sigma, cat. no. A8674); see REAGENT SETUP DOX (Sigma, cat. no. 44583); see REAGENT SETUP DETANONOate (cat. no. 82120); see REAGENT SETUP SOD-PEG (cat. no. S9549); see REAGENT SETUP MPMVECs RPMI (Invitrogen) medium supplemented with 10% FBS, essential amino acids, endothelial cell growth supplement and antibiotics DMEM (Invitrogen) supplemented with 10% FBS, nonessential amino acids, endothelial cell growth supplement and antibiotics Extracellular medium (ECM); see REAGENT SETUP Endothelial cell growth supplement (Upstate Biotech) Experimental imaging solution (ECM containing 0.25% BSA) containing sulfinpyrazone (Sigma) Annexin binding buffer (10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl 2 , pH 7.4) pEYFP-Mito (Clontech, BD Biosciences, cat. no. 6115-1) Basic Nucleofector kit for primary mammalian endothelial cells (Amaxa Biosystems, cat. no. VPI-1001) EQUIPMENT Tissue culture equipment 4 and −20 °C refrigerators Incubator (37 °C, 5% CO 2 (vol/vol)) Centrifuge Flow cytometer (FACSCalibur) (488 nm argon laser and 635 nm Red diode or 633 nm HeNe laser line, for excitation) Amaxa Nucleofector Device (Amaxa Biosystems) Appropriate imaging system, for example, Bio-Rad Radiance 2000 imaging system (Carl Zeiss MicroImaging) equipped with a Kr/Ar ion laser source and red diode laser source with excitation at 488, 568 and 638 nm Temperature-controlled stage (PDMI-2, open perfusion micro-incubator; Harvard Apparatus) Flow JO Version 8.5.2 (Three Stars Inc.) REAGENT SETUP FACS binding buffer Hank’s buffered salt solution (GIBCO, Invitrogen) containing 5 mM calcium chloride and magnesium chloride with 1% wt/vol BSA (cell culture tested; Sigma) MitoSOX Red Store at −20 °C. .. Antimycin A (Sigma, cat. no. A8674); see REAGENT SETUP DOX (Sigma, cat. no. 44583); see REAGENT SETUP DETANONOate (cat. no. 82120); see REAGENT SETUP SOD-PEG (cat. no. S9549); see REAGENT SETUP MPMVECs RPMI (Invitrogen) medium supplemented with 10% FBS, essential amino acids, endothelial cell growth supplement and antibiotics DMEM (Invitrogen) supplemented with 10% FBS, nonessential amino acids, endothelial cell growth supplement and antibiotics Extracellular medium (ECM); see REAGENT SETUP Endothelial cell growth supplement (Upstate Biotech) Experimental imaging solution (ECM containing 0.25% BSA) containing sulfinpyrazone (Sigma) Annexin binding buffer (10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl 2 , pH 7.4) pEYFP-Mito (Clontech, BD Biosciences, cat. no. 6115-1) Basic Nucleofector kit for primary mammalian endothelial cells (Amaxa Biosystems, cat. no. VPI-1001) EQUIPMENT Tissue culture equipment 4 and −20 °C refrigerators Incubator (37 °C, 5% CO 2 (vol/vol)) Centrifuge Flow cytometer (FACSCalibur) (488 nm argon laser and 635 nm Red diode or 633 nm HeNe laser line, for excitation) Amaxa Nucleofector Device (Amaxa Biosystems) Appropriate imaging system, for example, Bio-Rad Radiance 2000 imaging system (Carl Zeiss MicroImaging) equipped with a Kr/Ar ion laser source and red diode laser source with excitation at 488, 568 and 638 nm Temperature-controlled stage (PDMI-2, open perfusion micro-incubator; Harvard Apparatus) Flow JO Version 8.5.2 (Three Stars Inc.) REAGENT SETUP FACS binding buffer Hank’s buffered salt solution (GIBCO, Invitrogen) containing 5 mM calcium chloride and magnesium chloride with 1% wt/vol BSA (cell culture tested; Sigma) MitoSOX Red Store at −20 °C.
Binding Assay:Article Title: Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy
Article Snippet: .. Antimycin A (Sigma, cat. no. A8674); see REAGENT SETUP DOX (Sigma, cat. no. 44583); see REAGENT SETUP DETANONOate (cat. no. 82120); see REAGENT SETUP SOD-PEG (cat. no. S9549); see REAGENT SETUP MPMVECs RPMI (Invitrogen) medium supplemented with 10% FBS, essential amino acids, endothelial cell growth supplement and antibiotics DMEM (Invitrogen) supplemented with 10% FBS, nonessential amino acids, endothelial cell growth supplement and antibiotics Extracellular medium (ECM); see REAGENT SETUP Endothelial cell growth supplement (Upstate Biotech) Experimental imaging solution (ECM containing 0.25% BSA) containing sulfinpyrazone (Sigma) Annexin binding buffer (10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl 2 , pH 7.4) pEYFP-Mito (Clontech, BD Biosciences, cat. no. 6115-1) Basic Nucleofector kit for primary mammalian endothelial cells (Amaxa Biosystems, cat. no. VPI-1001) .. Antimycin A (Sigma, cat. no. A8674); see REAGENT SETUP DOX (Sigma, cat. no. 44583); see REAGENT SETUP DETANONOate (cat. no. 82120); see REAGENT SETUP SOD-PEG (cat. no. S9549); see REAGENT SETUP MPMVECs RPMI (Invitrogen) medium supplemented with 10% FBS, essential amino acids, endothelial cell growth supplement and antibiotics DMEM (Invitrogen) supplemented with 10% FBS, nonessential amino acids, endothelial cell growth supplement and antibiotics Extracellular medium (ECM); see REAGENT SETUP Endothelial cell growth supplement (Upstate Biotech) Experimental imaging solution (ECM containing 0.25% BSA) containing sulfinpyrazone (Sigma) Annexin binding buffer (10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl 2 , pH 7.4) pEYFP-Mito (Clontech, BD Biosciences, cat. no. 6115-1) Basic Nucleofector kit for primary mammalian endothelial cells (Amaxa Biosystems, cat. no. VPI-1001)
Article Title: Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy
Article Snippet: HCAECs growth medium (Cell Applications Inc., 212K-500) or an appropriate medium for growing cells of interest MitoSOX Red (Molecular Probes, Invitrogen, cat. no. {"type":"entrez-nucleotide","attrs":{"text":"M36008","term_id":"214108","term_text":"M36008"}} M36008 ) supplied in 50 μg vials; see REAGENT SETUP Sytox Green nucleic acid stain (Molecular Probes, Invitrogen, cat. no. S7020); see REAGENT SETUP Allophycocyanin (APC)-Annexin V (cat. no. {"type":"entrez-nucleotide","attrs":{"text":"A35110","term_id":"1926769","term_text":"A35110"}} A35110 ) and Alexa Fluor 647-Annexin V (cat. no. A23204; Molecular Probes, Invitrogen) (use directly from supplied stock). .. Antimycin A (Sigma, cat. no. A8674); see REAGENT SETUP DOX (Sigma, cat. no. 44583); see REAGENT SETUP DETANONOate (cat. no. 82120); see REAGENT SETUP SOD-PEG (cat. no. S9549); see REAGENT SETUP MPMVECs RPMI (Invitrogen) medium supplemented with 10% FBS, essential amino acids, endothelial cell growth supplement and antibiotics DMEM (Invitrogen) supplemented with 10% FBS, nonessential amino acids, endothelial cell growth supplement and antibiotics Extracellular medium (ECM); see REAGENT SETUP Endothelial cell growth supplement (Upstate Biotech) Experimental imaging solution (ECM containing 0.25% BSA) containing sulfinpyrazone (Sigma) Annexin binding buffer (10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl 2 , pH 7.4) pEYFP-Mito (Clontech, BD Biosciences, cat. no. 6115-1) Basic Nucleofector kit for primary mammalian endothelial cells (Amaxa Biosystems, cat. no. VPI-1001) EQUIPMENT Tissue culture equipment 4 and −20 °C refrigerators Incubator (37 °C, 5% CO 2 (vol/vol)) Centrifuge Flow cytometer (FACSCalibur) (488 nm argon laser and 635 nm Red diode or 633 nm HeNe laser line, for excitation) Amaxa Nucleofector Device (Amaxa Biosystems) Appropriate imaging system, for example, Bio-Rad Radiance 2000 imaging system (Carl Zeiss MicroImaging) equipped with a Kr/Ar ion laser source and red diode laser source with excitation at 488, 568 and 638 nm Temperature-controlled stage (PDMI-2, open perfusion micro-incubator; Harvard Apparatus) Flow JO Version 8.5.2 (Three Stars Inc.) REAGENT SETUP FACS binding buffer Hank’s buffered salt solution (GIBCO, Invitrogen) containing 5 mM calcium chloride and magnesium chloride with 1% wt/vol BSA (cell culture tested; Sigma) MitoSOX Red Store at −20 °C. .. Antimycin A (Sigma, cat. no. A8674); see REAGENT SETUP DOX (Sigma, cat. no. 44583); see REAGENT SETUP DETANONOate (cat. no. 82120); see REAGENT SETUP SOD-PEG (cat. no. S9549); see REAGENT SETUP MPMVECs RPMI (Invitrogen) medium supplemented with 10% FBS, essential amino acids, endothelial cell growth supplement and antibiotics DMEM (Invitrogen) supplemented with 10% FBS, nonessential amino acids, endothelial cell growth supplement and antibiotics Extracellular medium (ECM); see REAGENT SETUP Endothelial cell growth supplement (Upstate Biotech) Experimental imaging solution (ECM containing 0.25% BSA) containing sulfinpyrazone (Sigma) Annexin binding buffer (10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl 2 , pH 7.4) pEYFP-Mito (Clontech, BD Biosciences, cat. no. 6115-1) Basic Nucleofector kit for primary mammalian endothelial cells (Amaxa Biosystems, cat. no. VPI-1001) EQUIPMENT Tissue culture equipment 4 and −20 °C refrigerators Incubator (37 °C, 5% CO 2 (vol/vol)) Centrifuge Flow cytometer (FACSCalibur) (488 nm argon laser and 635 nm Red diode or 633 nm HeNe laser line, for excitation) Amaxa Nucleofector Device (Amaxa Biosystems) Appropriate imaging system, for example, Bio-Rad Radiance 2000 imaging system (Carl Zeiss MicroImaging) equipped with a Kr/Ar ion laser source and red diode laser source with excitation at 488, 568 and 638 nm Temperature-controlled stage (PDMI-2, open perfusion micro-incubator; Harvard Apparatus) Flow JO Version 8.5.2 (Three Stars Inc.) REAGENT SETUP FACS binding buffer Hank’s buffered salt solution (GIBCO, Invitrogen) containing 5 mM calcium chloride and magnesium chloride with 1% wt/vol BSA (cell culture tested; Sigma) MitoSOX Red Store at −20 °C.
Plasmid Preparation:Article Title: Nuclear envelope proteins Nesprin2 and LaminA regulate proliferation and apoptosis of vascular endothelial cells in response to shear stress
Article Snippet: Using a Basic Nucleofector Kit for Primary Mammalian Endothelial Cells (Amaxa, Germany), 3 μg of Nesprin2 or LaminA plasmid was transfected into 5 × 10 5 ECs with the program A-33 of Nucleofector II (Amaxa, Germany). .. Using a Basic Nucleofector Kit for Primary Mammalian Endothelial Cells (Amaxa, Germany), 3 μg of Nesprin2 or LaminA plasmid was transfected into 5 × 10 5 ECs with the program A-33 of Nucleofector II (Amaxa, Germany). .. Cell proliferation assay Cell proliferation was analyzed using an in situ BrdU kit (Roche Diagnostics, Germany) by fluorescence microscopy.
Transfection:Article Title: Nuclear envelope proteins Nesprin2 and LaminA regulate proliferation and apoptosis of vascular endothelial cells in response to shear stress
Article Snippet: Using a Basic Nucleofector Kit for Primary Mammalian Endothelial Cells (Amaxa, Germany), 3 μg of Nesprin2 or LaminA plasmid was transfected into 5 × 10 5 ECs with the program A-33 of Nucleofector II (Amaxa, Germany). .. Using a Basic Nucleofector Kit for Primary Mammalian Endothelial Cells (Amaxa, Germany), 3 μg of Nesprin2 or LaminA plasmid was transfected into 5 × 10 5 ECs with the program A-33 of Nucleofector II (Amaxa, Germany). .. Cell proliferation assay Cell proliferation was analyzed using an in situ BrdU kit (Roche Diagnostics, Germany) by fluorescence microscopy.
Article Title: IL-1β suppression of VE-cadherin transcription underlies sepsis-induced inflammatory lung injury
Article Snippet: The scrambled siRNA duplex controls were designed and supplied by Dharmacon. .. Transfection of siRNA duplexes in cells was performed according to the manufacturer’s instructions for the Basic Nucleofector Kit for Primary Endothelial Cells (Amaxa Biosystem). ..
Flow Cytometry:Article Title: Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy
Article Snippet: HCAECs growth medium (Cell Applications Inc., 212K-500) or an appropriate medium for growing cells of interest MitoSOX Red (Molecular Probes, Invitrogen, cat. no. {"type":"entrez-nucleotide","attrs":{"text":"M36008","term_id":"214108","term_text":"M36008"}} M36008 ) supplied in 50 μg vials; see REAGENT SETUP Sytox Green nucleic acid stain (Molecular Probes, Invitrogen, cat. no. S7020); see REAGENT SETUP Allophycocyanin (APC)-Annexin V (cat. no. {"type":"entrez-nucleotide","attrs":{"text":"A35110","term_id":"1926769","term_text":"A35110"}} A35110 ) and Alexa Fluor 647-Annexin V (cat. no. A23204; Molecular Probes, Invitrogen) (use directly from supplied stock). .. Antimycin A (Sigma, cat. no. A8674); see REAGENT SETUP DOX (Sigma, cat. no. 44583); see REAGENT SETUP DETANONOate (cat. no. 82120); see REAGENT SETUP SOD-PEG (cat. no. S9549); see REAGENT SETUP MPMVECs RPMI (Invitrogen) medium supplemented with 10% FBS, essential amino acids, endothelial cell growth supplement and antibiotics DMEM (Invitrogen) supplemented with 10% FBS, nonessential amino acids, endothelial cell growth supplement and antibiotics Extracellular medium (ECM); see REAGENT SETUP Endothelial cell growth supplement (Upstate Biotech) Experimental imaging solution (ECM containing 0.25% BSA) containing sulfinpyrazone (Sigma) Annexin binding buffer (10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl 2 , pH 7.4) pEYFP-Mito (Clontech, BD Biosciences, cat. no. 6115-1) Basic Nucleofector kit for primary mammalian endothelial cells (Amaxa Biosystems, cat. no. VPI-1001) EQUIPMENT Tissue culture equipment 4 and −20 °C refrigerators Incubator (37 °C, 5% CO 2 (vol/vol)) Centrifuge Flow cytometer (FACSCalibur) (488 nm argon laser and 635 nm Red diode or 633 nm HeNe laser line, for excitation) Amaxa Nucleofector Device (Amaxa Biosystems) Appropriate imaging system, for example, Bio-Rad Radiance 2000 imaging system (Carl Zeiss MicroImaging) equipped with a Kr/Ar ion laser source and red diode laser source with excitation at 488, 568 and 638 nm Temperature-controlled stage (PDMI-2, open perfusion micro-incubator; Harvard Apparatus) Flow JO Version 8.5.2 (Three Stars Inc.) REAGENT SETUP FACS binding buffer Hank’s buffered salt solution (GIBCO, Invitrogen) containing 5 mM calcium chloride and magnesium chloride with 1% wt/vol BSA (cell culture tested; Sigma) MitoSOX Red Store at −20 °C. .. Antimycin A (Sigma, cat. no. A8674); see REAGENT SETUP DOX (Sigma, cat. no. 44583); see REAGENT SETUP DETANONOate (cat. no. 82120); see REAGENT SETUP SOD-PEG (cat. no. S9549); see REAGENT SETUP MPMVECs RPMI (Invitrogen) medium supplemented with 10% FBS, essential amino acids, endothelial cell growth supplement and antibiotics DMEM (Invitrogen) supplemented with 10% FBS, nonessential amino acids, endothelial cell growth supplement and antibiotics Extracellular medium (ECM); see REAGENT SETUP Endothelial cell growth supplement (Upstate Biotech) Experimental imaging solution (ECM containing 0.25% BSA) containing sulfinpyrazone (Sigma) Annexin binding buffer (10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl 2 , pH 7.4) pEYFP-Mito (Clontech, BD Biosciences, cat. no. 6115-1) Basic Nucleofector kit for primary mammalian endothelial cells (Amaxa Biosystems, cat. no. VPI-1001) EQUIPMENT Tissue culture equipment 4 and −20 °C refrigerators Incubator (37 °C, 5% CO 2 (vol/vol)) Centrifuge Flow cytometer (FACSCalibur) (488 nm argon laser and 635 nm Red diode or 633 nm HeNe laser line, for excitation) Amaxa Nucleofector Device (Amaxa Biosystems) Appropriate imaging system, for example, Bio-Rad Radiance 2000 imaging system (Carl Zeiss MicroImaging) equipped with a Kr/Ar ion laser source and red diode laser source with excitation at 488, 568 and 638 nm Temperature-controlled stage (PDMI-2, open perfusion micro-incubator; Harvard Apparatus) Flow JO Version 8.5.2 (Three Stars Inc.) REAGENT SETUP FACS binding buffer Hank’s buffered salt solution (GIBCO, Invitrogen) containing 5 mM calcium chloride and magnesium chloride with 1% wt/vol BSA (cell culture tested; Sigma) MitoSOX Red Store at −20 °C.
FACS:Article Title: Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy
Article Snippet: HCAECs growth medium (Cell Applications Inc., 212K-500) or an appropriate medium for growing cells of interest MitoSOX Red (Molecular Probes, Invitrogen, cat. no. {"type":"entrez-nucleotide","attrs":{"text":"M36008","term_id":"214108","term_text":"M36008"}} M36008 ) supplied in 50 μg vials; see REAGENT SETUP Sytox Green nucleic acid stain (Molecular Probes, Invitrogen, cat. no. S7020); see REAGENT SETUP Allophycocyanin (APC)-Annexin V (cat. no. {"type":"entrez-nucleotide","attrs":{"text":"A35110","term_id":"1926769","term_text":"A35110"}} A35110 ) and Alexa Fluor 647-Annexin V (cat. no. A23204; Molecular Probes, Invitrogen) (use directly from supplied stock). .. Antimycin A (Sigma, cat. no. A8674); see REAGENT SETUP DOX (Sigma, cat. no. 44583); see REAGENT SETUP DETANONOate (cat. no. 82120); see REAGENT SETUP SOD-PEG (cat. no. S9549); see REAGENT SETUP MPMVECs RPMI (Invitrogen) medium supplemented with 10% FBS, essential amino acids, endothelial cell growth supplement and antibiotics DMEM (Invitrogen) supplemented with 10% FBS, nonessential amino acids, endothelial cell growth supplement and antibiotics Extracellular medium (ECM); see REAGENT SETUP Endothelial cell growth supplement (Upstate Biotech) Experimental imaging solution (ECM containing 0.25% BSA) containing sulfinpyrazone (Sigma) Annexin binding buffer (10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl 2 , pH 7.4) pEYFP-Mito (Clontech, BD Biosciences, cat. no. 6115-1) Basic Nucleofector kit for primary mammalian endothelial cells (Amaxa Biosystems, cat. no. VPI-1001) EQUIPMENT Tissue culture equipment 4 and −20 °C refrigerators Incubator (37 °C, 5% CO 2 (vol/vol)) Centrifuge Flow cytometer (FACSCalibur) (488 nm argon laser and 635 nm Red diode or 633 nm HeNe laser line, for excitation) Amaxa Nucleofector Device (Amaxa Biosystems) Appropriate imaging system, for example, Bio-Rad Radiance 2000 imaging system (Carl Zeiss MicroImaging) equipped with a Kr/Ar ion laser source and red diode laser source with excitation at 488, 568 and 638 nm Temperature-controlled stage (PDMI-2, open perfusion micro-incubator; Harvard Apparatus) Flow JO Version 8.5.2 (Three Stars Inc.) REAGENT SETUP FACS binding buffer Hank’s buffered salt solution (GIBCO, Invitrogen) containing 5 mM calcium chloride and magnesium chloride with 1% wt/vol BSA (cell culture tested; Sigma) MitoSOX Red Store at −20 °C. .. Antimycin A (Sigma, cat. no. A8674); see REAGENT SETUP DOX (Sigma, cat. no. 44583); see REAGENT SETUP DETANONOate (cat. no. 82120); see REAGENT SETUP SOD-PEG (cat. no. S9549); see REAGENT SETUP MPMVECs RPMI (Invitrogen) medium supplemented with 10% FBS, essential amino acids, endothelial cell growth supplement and antibiotics DMEM (Invitrogen) supplemented with 10% FBS, nonessential amino acids, endothelial cell growth supplement and antibiotics Extracellular medium (ECM); see REAGENT SETUP Endothelial cell growth supplement (Upstate Biotech) Experimental imaging solution (ECM containing 0.25% BSA) containing sulfinpyrazone (Sigma) Annexin binding buffer (10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl 2 , pH 7.4) pEYFP-Mito (Clontech, BD Biosciences, cat. no. 6115-1) Basic Nucleofector kit for primary mammalian endothelial cells (Amaxa Biosystems, cat. no. VPI-1001) EQUIPMENT Tissue culture equipment 4 and −20 °C refrigerators Incubator (37 °C, 5% CO 2 (vol/vol)) Centrifuge Flow cytometer (FACSCalibur) (488 nm argon laser and 635 nm Red diode or 633 nm HeNe laser line, for excitation) Amaxa Nucleofector Device (Amaxa Biosystems) Appropriate imaging system, for example, Bio-Rad Radiance 2000 imaging system (Carl Zeiss MicroImaging) equipped with a Kr/Ar ion laser source and red diode laser source with excitation at 488, 568 and 638 nm Temperature-controlled stage (PDMI-2, open perfusion micro-incubator; Harvard Apparatus) Flow JO Version 8.5.2 (Three Stars Inc.) REAGENT SETUP FACS binding buffer Hank’s buffered salt solution (GIBCO, Invitrogen) containing 5 mM calcium chloride and magnesium chloride with 1% wt/vol BSA (cell culture tested; Sigma) MitoSOX Red Store at −20 °C.
Cell Culture:Article Title: Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy
Article Snippet: HCAECs growth medium (Cell Applications Inc., 212K-500) or an appropriate medium for growing cells of interest MitoSOX Red (Molecular Probes, Invitrogen, cat. no. {"type":"entrez-nucleotide","attrs":{"text":"M36008","term_id":"214108","term_text":"M36008"}} M36008 ) supplied in 50 μg vials; see REAGENT SETUP Sytox Green nucleic acid stain (Molecular Probes, Invitrogen, cat. no. S7020); see REAGENT SETUP Allophycocyanin (APC)-Annexin V (cat. no. {"type":"entrez-nucleotide","attrs":{"text":"A35110","term_id":"1926769","term_text":"A35110"}} A35110 ) and Alexa Fluor 647-Annexin V (cat. no. A23204; Molecular Probes, Invitrogen) (use directly from supplied stock). .. Antimycin A (Sigma, cat. no. A8674); see REAGENT SETUP DOX (Sigma, cat. no. 44583); see REAGENT SETUP DETANONOate (cat. no. 82120); see REAGENT SETUP SOD-PEG (cat. no. S9549); see REAGENT SETUP MPMVECs RPMI (Invitrogen) medium supplemented with 10% FBS, essential amino acids, endothelial cell growth supplement and antibiotics DMEM (Invitrogen) supplemented with 10% FBS, nonessential amino acids, endothelial cell growth supplement and antibiotics Extracellular medium (ECM); see REAGENT SETUP Endothelial cell growth supplement (Upstate Biotech) Experimental imaging solution (ECM containing 0.25% BSA) containing sulfinpyrazone (Sigma) Annexin binding buffer (10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl 2 , pH 7.4) pEYFP-Mito (Clontech, BD Biosciences, cat. no. 6115-1) Basic Nucleofector kit for primary mammalian endothelial cells (Amaxa Biosystems, cat. no. VPI-1001) EQUIPMENT Tissue culture equipment 4 and −20 °C refrigerators Incubator (37 °C, 5% CO 2 (vol/vol)) Centrifuge Flow cytometer (FACSCalibur) (488 nm argon laser and 635 nm Red diode or 633 nm HeNe laser line, for excitation) Amaxa Nucleofector Device (Amaxa Biosystems) Appropriate imaging system, for example, Bio-Rad Radiance 2000 imaging system (Carl Zeiss MicroImaging) equipped with a Kr/Ar ion laser source and red diode laser source with excitation at 488, 568 and 638 nm Temperature-controlled stage (PDMI-2, open perfusion micro-incubator; Harvard Apparatus) Flow JO Version 8.5.2 (Three Stars Inc.) REAGENT SETUP FACS binding buffer Hank’s buffered salt solution (GIBCO, Invitrogen) containing 5 mM calcium chloride and magnesium chloride with 1% wt/vol BSA (cell culture tested; Sigma) MitoSOX Red Store at −20 °C. .. Antimycin A (Sigma, cat. no. A8674); see REAGENT SETUP DOX (Sigma, cat. no. 44583); see REAGENT SETUP DETANONOate (cat. no. 82120); see REAGENT SETUP SOD-PEG (cat. no. S9549); see REAGENT SETUP MPMVECs RPMI (Invitrogen) medium supplemented with 10% FBS, essential amino acids, endothelial cell growth supplement and antibiotics DMEM (Invitrogen) supplemented with 10% FBS, nonessential amino acids, endothelial cell growth supplement and antibiotics Extracellular medium (ECM); see REAGENT SETUP Endothelial cell growth supplement (Upstate Biotech) Experimental imaging solution (ECM containing 0.25% BSA) containing sulfinpyrazone (Sigma) Annexin binding buffer (10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl 2 , pH 7.4) pEYFP-Mito (Clontech, BD Biosciences, cat. no. 6115-1) Basic Nucleofector kit for primary mammalian endothelial cells (Amaxa Biosystems, cat. no. VPI-1001) EQUIPMENT Tissue culture equipment 4 and −20 °C refrigerators Incubator (37 °C, 5% CO 2 (vol/vol)) Centrifuge Flow cytometer (FACSCalibur) (488 nm argon laser and 635 nm Red diode or 633 nm HeNe laser line, for excitation) Amaxa Nucleofector Device (Amaxa Biosystems) Appropriate imaging system, for example, Bio-Rad Radiance 2000 imaging system (Carl Zeiss MicroImaging) equipped with a Kr/Ar ion laser source and red diode laser source with excitation at 488, 568 and 638 nm Temperature-controlled stage (PDMI-2, open perfusion micro-incubator; Harvard Apparatus) Flow JO Version 8.5.2 (Three Stars Inc.) REAGENT SETUP FACS binding buffer Hank’s buffered salt solution (GIBCO, Invitrogen) containing 5 mM calcium chloride and magnesium chloride with 1% wt/vol BSA (cell culture tested; Sigma) MitoSOX Red Store at −20 °C.
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