The plasmid, miTarget miRNA 3′ Target Clone (HmiT001838‐MT01; GeneCopoeia) was used in the luciferase reporter assay. GFP‐SAS cells were seeded in 96‐well plates and after 24 hours they were cotransfected with LNA‐miR‐361‐3p (20 nmol/L) and the OSR2 3′‐UTR plasmid (50 ng/well) complexed with Lipofectamine LTX Reagent (Thermo Fisher Scientific), and then incubated for 10 hours. Firefly and Renilla luciferase activities were measured sequentially using the Dual‐Glo Luciferase Assay System (Promega). Results were expressed as relative luciferase activity units measured using a Wallac 1420 ARVO MX/Light (PerkinElmer).
Wallac 1420 arvo mx light
Manufactured by PerkinElmer
Sourced in United States
The Wallac 1420 ARVO MX/Light is a multimode microplate reader capable of detecting various luminescent, fluorescent, and absorbance signals. It is designed to perform a range of assays and applications in life science research and drug discovery.
Automatically generated - may contain errors
Lab products found in correlation
Dihydroethidium, by Merck Group (4 mentions)
Dual glo luciferase assay system, by Promega (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Lipofectamine ltx reagent, by Thermo Fisher Scientific (1 mentions)
Telotaggg telomerase pcr elisa plus kit, by Roche (1 mentions)
Cell atp assay reagent, by Fujifilm (1 mentions)
9 protocols using wallac 1420 arvo mx light
1
Regulation of OSR2 by miR-361-3p
2
Luciferase Reporter Assay for miRNA Regulation
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The plasmid, miTarget miRNA 3′ Target Clone (Cat. #HmiT020443-MT01, GeneCopoeia, Rockville, MD, USA) was specifically synthesized and used for a luciferase reporter assay. These plasmids contain firefly luciferase that is fused to the 3′-untranslated region (UTR) of human MAGT1 and Renilla luciferase, which functions as a tracking gene. GFP-SAS cells were seeded in 96-well plates. After 24 h, the cells were cotransfected with miR-1289 mimics or miR-NT (100 nM) and a plasmid containing MAGT1 3′-UTR (10 ng/well) complexed with Lipofectamine 3000 (Thermo Fisher Scientific), and incubated for 24 h. Firefly and Renilla luciferase activities were measured sequentially using the Dual-Glo®® luciferase Assay System (Promega, Madison, WI, USA). The results are expressed as relative luciferase activity units using Wallac 1420 ARVO MX/Light (PerkinElmer, Waltham, MA, USA). The activities were normalized to Renilla luciferase activity.
3
Quantifying Myocardial Superoxide Production
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Nicotinamide adenine dinucleotide phosphate (NADPH)-dependent superoxide production by homogenates prepared from freshly frozen LV tissue was measured with an assay based on lucigenin-enhanced chemiluminescence as described previously.16 ) The chemiluminescence signal was sampled every minute for 10 min with a microplate reader (WALLAC 1420 ARVO MX/Light; Perkin-Elmer, Waltham, MA), and the respective background counts were subtracted from experimental values. Superoxide production in tissue sections was examined as described17 ) with the use of dihydroethidium (Sigma), which is rapidly oxidized by superoxide to yield fluorescent ethidium. Sections were examined with a fluorescence microscope equipped with a 585-nm long-pass filter. As a negative control, sections were incubated with superoxide dismutase (300 U/mL) before staining with dihydroethidium; such treatment prevented the generation of fluorescence signals (data not shown).
4
Measuring Cardiac Oxidative Stress
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Nicotinamide adenine dinucleotide phosphate (NADPH)‐dependent superoxide production by homogenates prepared from freshly frozen LV tissue was measured with an assay based on lucigenin‐enhanced chemiluminescence as described previously.21 (link) The chemiluminescence signal was sampled every minute for 10 minutes with a microplate reader (WALLAC 1420 ARVO MX/Light; Perkin‐Elmer, Waltham, MA), and the respective background counts were subtracted from experimental values. Sections stained with dihydroethidium (Sigma) were examined with a fluorescence microscope equipped with a 585‐nm long‐pass filter. As a negative control, sections were incubated with superoxide dismutase (300 U/mL) before staining with dihydroethidium; such treatment prevented the generation of fluorescence signals (data not shown). The average of dihydroethidium fluorescence intensity values was calculated with NIH ImageJ software.
5
Measuring Superoxide Production in Cardiac Tissue
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Nicotinamide adenine dinucleotide phosphate (NADPH)-dependent superoxide production by homogenates prepared from freshly frozen LV tissue was measured with an assay based on lucigenin-enhanced chemiluminescence, as described previously.18 ) The chemiluminescence signal was sampled every minute for 10 min with a microplate reader (Wallac 1420 ARVO MX/Light; Perkin-Elmer, Waltham, MA), and the respective background counts were subtracted from experimental values. Superoxide production in tissue sections was examined by staining with dihydroethidium (Sigma, St. Louis, MO) as described previously.17 , 29 )
6
Quantifying Superoxide Production in Cardiac Tissue
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Nicotinamide adenine dinucleotide phosphate (NADPH)-dependent superoxide production by homogenates of freshly frozen LV tissue was measured with an assay based on lucigenin-enhanced chemiluminescence as described previously.12 ) The chemiluminescence signal was sampled every minute for 10 min with a microplate reader (Wallac 1420 ARVO MX/Light; Perkin-Elmer, Waltham, MA, USA), and the respective background counts were subtracted from experimental values. Superoxide production in tissue sections was examined by staining with dihydroethidium (Sigma, St. Louis, MO, USA) as described.13 ) Dihydroethidium is rapidly oxidized by superoxide to yield fluorescent ethidium, and the sections were examined with a fluorescence microscope equipped with a 585-nm long-pass filter. As a negative control, sections were incubated with superoxide dismutase (300 U/mL) before staining with dihydroethidium; such treatment prevented the generation of fluorescence signals (data not shown). The average of dihydroethidium fluorescence intensity values was calculated with the use of NIH Image software (ImageJ).
7
Telomerase Activity Quantification by ELISA
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Telomerase activity was measured with the use of a Telo TAGGG Telomerase PCR ELISAPLUS Kit (Roche). Total protein (1 μg) isolated from LV tissue and quantitated by ultraviolet absorption spectrometry was mixed with a biotinylated telomerase substrate, an optimized anchor-primer, nucleotides, Taq DNA polymerase, and an internal standard, and the mixture was incubated first for 20 min at 25°C to allow primer elongation and then for 5 min at 94°C to inactivate telomerase. After 30 cycles of amplification (30 s at 94°C, 30 s at 50°C, and 90 s at 72°C), the PCR products were divided into two portions, denatured, and subjected to hybridization separately with digoxigenin-labeled detection probes specific either for telomeric repeats or for the internal standard. The resulting products were immobilized on a streptavidin-coated microplate and detected with horseradish peroxidase–conjugated antibodies to digoxigenin and a peroxidase substrate. The absorbance of the samples was measured with the use of a microplate reader (Wallac 1420 ARVO MX/Light, Perkin-Elmer).
8
Monomer Effects on MC3T3-E1 Proliferation
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The MC3T3-E1 cells were seeded into the wells of a 96-well tissue culture plate at 2.0×10 4 cells/well and cultured for 12 h in a humidified 5% CO2 incubator at 37°C. The media was replaced with fresh growth media containing 4-MET or MMA. The concentrations of each monomer tested (5 or 10 µg/mL of 4-MET, and 5, 10, or 25 µg/mL of MMA) were decided based on the results of a previously-conducted HPLC analysis of monomer release. FGF-2 (Fiblast; Kaken Pharmaceutical, Tokyo, Japan) was added to each well at a final concentration of 5 ng/mL and the culture was continued for another 24 h. Cells cultured without FGF-2 were used as controls.
Cell proliferation was determined by MTT assay. Briefly, 20 µL of 3-(4,5-dimethylthiazoly1-2)-2,5diphenyl-tetrazolium bromide solution (MTT; Sigma Chemical, St. Louis, MO, USA) was added to each well and the plates were incubated for 4 h at 37°C. After removing the media, 110 µL of 2% SDS/0.01 N HCl (Nacalai Tesque, Kyoto, Japan) was added to each well. The absorbance at 570 nm was measured using a microplate reader (Wallac 1420 ARVO Mx/Light; PerkinElmer, Waltham, MA, USA). The experiments were repeated three times independently.
Cell proliferation was determined by MTT assay. Briefly, 20 µL of 3-(4,5-dimethylthiazoly1-2)-2,5diphenyl-tetrazolium bromide solution (MTT; Sigma Chemical, St. Louis, MO, USA) was added to each well and the plates were incubated for 4 h at 37°C. After removing the media, 110 µL of 2% SDS/0.01 N HCl (Nacalai Tesque, Kyoto, Japan) was added to each well. The absorbance at 570 nm was measured using a microplate reader (Wallac 1420 ARVO Mx/Light; PerkinElmer, Waltham, MA, USA). The experiments were repeated three times independently.
9
Ethanol-Induced Cardiomyocyte ATP Depletion
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Cardiomyocytes cultured in a 96-well plastic plate were exposed to ethanol in 100 μL of 21 mM HEPESbuffered serum-free medium. Cellular ATP content was assayed by detecting luciferin-luciferase chemiluminescence reaction. Twenty-four hours after ethanol exposure was started, the plate was placed at room temperature for 30 minutes. To each well 100 μL of "Cell" ATP Assay Reagent (Wako Pure Chemical Industries, Ltd., Osaka, Japan) containing D-luciferin and luciferase was added and mixed well in dark on a microplate shaker for 1 minute.
After the plate had been incubated in the measuring chamber of a plate reader (Wallac 1420 ARVOMX! Light, PerkinElmer Inc., Turku, Finland) for 10 minutes at 23°C, chemiluminescence was measured at 23°C.
After the plate had been incubated in the measuring chamber of a plate reader (Wallac 1420 ARVOMX! Light, PerkinElmer Inc., Turku, Finland) for 10 minutes at 23°C, chemiluminescence was measured at 23°C.
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