Cellular ROS levels were ascertained using the cell permeable probe CM-H2DCFDA (5-(and-6)-chloromethyl-2’7’ dichlorodihydrofluorescein diacetate acetyl ester) from Invitrogen (Grand Island, NY). CM-H2DCFDA is non-fluorescent until cleavage of the acetyl groups by intracellular esterases and oxidation that transpires within the cell. Following treatment of the cells for 12 hours with DIM-C-pPhOH (+/− 5mM GSH 3 h prior to treatment) for 72 hours, cells plated on a 6-well culture plate were trypsinized, neutralized, then loaded with 10μM of probe for 20 min, washed once with serum free medium, and then ROS was measured by flow cytometry using Accuri’s C6 Flow Cytometer (Accuri, Ann Arbor, MI).
Accuri s c6 flow cytometer
Manufactured by BD
Sourced in United States
The Accuri C6 Flow Cytometer is a compact and versatile instrument designed for automated cell analysis. It utilizes flow cytometry technology to rapidly measure and analyze the physical and fluorescent characteristics of cells or particles suspended in a fluid stream.
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2 protocols using accuri s c6 flow cytometer
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Measuring Cellular Reactive Oxygen Species
2
Quantifying Cellular Reactive Oxygen Levels
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Cellular ROS levels were ascertained using the cell permeable probe CM-H 2 DCFDA (5-(and-6)-chloromethyl-2 0 7 0 -dichlorodihydrofluorescein diacetate acetyl ester) from Invitrogen. Following treatment of the cells for 12 or 24 h with DIM-C-pPHCO 2 Me or siNR4A1 for 72 h, cells that were plated on a six-well culture plate were trypsinized, neutralized, then loaded with 10 mM of probe for 20 min, and washed once with serum free medium. ROS was then measured by flow cytometry using Accuri's C6 Flow Cytometer (Accuri, Ann Arbor, MI, USA).
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