Production of reactive oxygen species (ROS) in mouse tissues was measured in vitro using a previously described protocol134 (link) with a few modifications. All chemicals used in this assay were purchased from Merck (Darmstadt, Germany). We mixed 45 µl protein samples (30 µg protein) with 50 µl ice-cold 2x Locke’s buffer (308 mM NaCl, 11.2 mM KCl, 7.2 mM NaHCO3, 4 mM CaCl2, 20 mM D-glucose, 10 mM HEPES (pH7.4)) and loaded this mixture onto a black 96-well plate (Sarstedt, Nümbrecht, Germany). We then added 5 µl 200 µM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and the reaction mixture was incubated in the dark on an orbital shaker for 30 min at room temperature and 50 rpm. Fluorescent intensities (excitation 485 nm, emission 530 nm) were acquired using a Tecan Infinite M200 Pro plate reader (Tecan, Männedorf, Switzerland).
96 well black plate
Manufactured by Sarstedt
Sourced in Germany
The 96-well black plate is a laboratory equipment designed for various applications that require a high-contrast, opaque background. It is commonly used in fluorescence-based assays and other experimental procedures that benefit from a dark environment to minimize background interference and enhance signal detection.
Automatically generated - may contain errors
Lab products found in correlation
Victor3v multilabel plate reader, by PerkinElmer (3 mentions)
Proteasome glo 3 substrate system, by Promega (2 mentions)
Quantikine elisa, by R&D Systems (2 mentions)
Basal dmem, by Lonza (2 mentions)
Amicon ultra centrifugal filters 100 kb, by Merck Group (2 mentions)
Infinite m200 pro, by Tecan (1 mentions)
Infinite m200 pro plate reader, by Tecan (1 mentions)
5 protocols using 96 well black plate
1
Quantifying ROS in Mouse Tissues
2
In Vitro 20S Proteasome Activity Assay
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The 20S proteasome activity was assessed in vitro using a protocol published elsewhere133 (link). Developing buffer (50 mM Tris (pH7.5), 150 mM NaCl, 5 mM MgCl2) containing 30 µg protein in a total volume of 94 µl was loaded onto a black 96-well plate (Sarstedt, Nümbrecht, Germany). Two µl 50 mM ATP-Mg2+ (Merck, Darmstadt, Germany), 2 µl 5 mM Suc-LLVY-AMC substrate (Cayman Chemical, Ann Arbor, MI, USA) and 2 µl 1% SDS (Carl Roth, Karlsruhe, Germany) were added immediately prior to commencing the experiment. Reaction suspensions were incubated in the dark at 37 °C for 30 min and fluorescent signals (excitation 380 nm, emission 460 nm) were acquired using a Tecan Infinite M200 Pro plate reader (Tecan, Männedorf, Switzerland).
3
Proteasome Activity Assay in Cells
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Cells were seeded in a 96- well black plate (Sarstedt, Nümbrecht, Germany) at 5000 cells/well in 3 replicates until confluence. The media was then changed to differentiation medium. Cells were treated with the corresponding drug for the indicated time. The CT-L, C-T and T-L activity was assayed by chemiluminiscence using the Proteasome-Glo™ 3-substrate System cell based assay (Promega, Madison, WI, USA) and the plate was read using Victor 3v Multilabel Plate Reader (Perkin Elmer, Waltham, MA, USA).
4
Proteasome Activity and TSP-1 in Myoblasts
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Cells were seeded in a 96-well black plate (Sarstedt, Nümbrecht, Germany) at 5000 cells/well in three replicates until con uence. The media was then changed to differentiation medium. Cells were treated with the corresponding drug for the indicated time. CT-L, C-T and T-L activity was assayed by chemiluminiscence using the Proteasome-Glo™ 3-Substrate System cell based assay (Promega, Madison, WI, USA ) and the plate was read using Victor 3v Multilabel Plate Reader (Perkin Elmer, Waltham, MA, USA).
Thrombospondin-1 Enzyme-Linked Immunosorbent Assay (ELISA)
Immortalized myoblasts from the dysferlinopathy patient were seeded at 5000 cells/cm 2 and expanded until con uence. The media was then changed to differentiation medium to form myotubes. After treatment with proteasome inhibitors together with or without EB1089, the media was removed and added 1 ml of basal DMEM (Lonza) was added to the culture for 24 hours. Cell culture supernatants were concentrated using Amicon Ultra Centrifugal Filters 100 kb (Merck Millipore, Darmstadt, Germany). TSP-1 present in the culture media was detected using the human TSP-1 Immunoassay (Quanti-Kine ELISA, R&D Systems, Minneapolis, MN), following the manufacturer's instructions. The detection limit of the assay was 0.355 ng/mL.
Thrombospondin-1 Enzyme-Linked Immunosorbent Assay (ELISA)
Immortalized myoblasts from the dysferlinopathy patient were seeded at 5000 cells/cm 2 and expanded until con uence. The media was then changed to differentiation medium to form myotubes. After treatment with proteasome inhibitors together with or without EB1089, the media was removed and added 1 ml of basal DMEM (Lonza) was added to the culture for 24 hours. Cell culture supernatants were concentrated using Amicon Ultra Centrifugal Filters 100 kb (Merck Millipore, Darmstadt, Germany). TSP-1 present in the culture media was detected using the human TSP-1 Immunoassay (Quanti-Kine ELISA, R&D Systems, Minneapolis, MN), following the manufacturer's instructions. The detection limit of the assay was 0.355 ng/mL.
5
Proteasome Activity and Thrombospondin-1 Measurement
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Cells were seeded in a 96-well black plate (Sarstedt, Nümbrecht, Germany) at 5000 cells/well in 3 replicates until con uence. The media was then changed to differentiation medium. Cells were treated with the corresponding drug for the indicated time. The CT-L, C-T and T-L activity was assayed by chemiluminiscence using the Proteasome-Glo™ 3-substrate System cell based assay (Promega, Madison, WI, USA ) and the plate was read using Victor 3v Multilabel Plate Reader (Perkin Elmer, Waltham, MA, USA).
Thrombospondin-1 Enzyme-Linked Immunosorbent Assay (ELISA)
Immortalized myoblasts from the dysferlinopathy patient were seeded at 5000 cells/cm 2 and expanded until con uence. The media was then changed to differentiation medium to form myotubes. After treatment with proteasome inhibitors together with or without EB1089, we removed the media and added 1 ml of basal DMEM (Lonza) to the culture for 24 hours. Cell culture supernatants were concentrated using Amicon Ultra Centrifugal Filters 100 kb (Merck Millipore, Darmstadt, Germany). TSP-1 present in the culture media was detected using the human TSP-1 Immunoassay (Quanti-Kine ELISA, R&D Systems, Minneapolis, MN), following the manufacturer's instructions. The detection limit of the assay was 0.355 ng/mL.
Thrombospondin-1 Enzyme-Linked Immunosorbent Assay (ELISA)
Immortalized myoblasts from the dysferlinopathy patient were seeded at 5000 cells/cm 2 and expanded until con uence. The media was then changed to differentiation medium to form myotubes. After treatment with proteasome inhibitors together with or without EB1089, we removed the media and added 1 ml of basal DMEM (Lonza) to the culture for 24 hours. Cell culture supernatants were concentrated using Amicon Ultra Centrifugal Filters 100 kb (Merck Millipore, Darmstadt, Germany). TSP-1 present in the culture media was detected using the human TSP-1 Immunoassay (Quanti-Kine ELISA, R&D Systems, Minneapolis, MN), following the manufacturer's instructions. The detection limit of the assay was 0.355 ng/mL.
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