To detect intracellular ROS, cells were incubated with 20 µM DCF-DA solution (Abcam, Cambridge, UK) for 30 min at 37°C and then washed with DPBS. Mitochondrial superoxide activity was detected using fluorescent MitoSOX (Invitrogen). Cells were incubated in complete medium containing 5 µM MitoSOX for 30 min at 37°C and then washed with DPBS. To measure the ΔΨm, cells were incubated for 30 min at 37°C with 10 µg/mL of JC-1 (Invitrogen) and then washed with DPBS. Fluorescence was assessed in a CytoFLEX flow cytometer (Beckman Coulter).
Dcf da solution
Manufactured by Abcam
Sourced in United Kingdom
DCF-DA solution is a fluorescent dye used to detect and measure reactive oxygen species (ROS) in biological systems. It functions as a probe, undergoing chemical transformation upon reaction with ROS, resulting in the emission of fluorescent signal that can be quantified.
Automatically generated - may contain errors
Lab products found in correlation
Mitosox, by Thermo Fisher Scientific (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Fluostar optima, by BMG Labtech (1 mentions)
Phenol red free dmem, by Thermo Fisher Scientific (1 mentions)
Ang 2, by Merck Group (1 mentions)
Tert butyl hydrogen peroxide tbhp, by Abcam (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
880 airyscan confocal microscope, by Zeiss (1 mentions)
5 protocols using dcf da solution
1
Intracellular ROS and Mitochondrial Assays
2
Oxidative Stress Response to Angiotensin II
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Cells were seeded in a dark-wall, clear-bottom, 96-well microplate (Costar®; Corning Inc., New York, NY, USA) at a density of 20,000 cells/well. 100 µL of DCFDA solution (10 µmol/L in HBSS) (Abcam) were added to each well and cells were stained for 45 min at 37 °C in the dark. Subsequently, cells were washed and treated with 1 µmol/L AngII (Sigma Aldrich) in the presence of 800 µg/mL nHDL with or without inhibitors for 6 h. A total of 200 µmol/L tert-butyl hydrogen peroxide (TBHP; Abcam) was used as positive control. All treatment compounds were diluted in phenol red free DMEM (Gibco, Thermo Fisher Scientific) without supplements. Fluorescent intensity of oxidized dichlorofluorescein (DCF) was measured at Ex/Em = 485/535 in end point mode (FLUOstar Optima; BMG Labtech, Offenburg, Germany).
3
Evaluating Oxidative Stress in HK2 Cells
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The HK2 cells were seeded at 5 × 105 cells/well on 6-well plates. The cells were pretreated with EVs (1 × 108 particles/mL) for 2 h, and damaged with hydrogen peroxide (H2O2, 0.5 mM, Sigma-Aldrich, St. Louis, MO, USA) to upregulate ROS levels for 2 h. The DCF-DA solution (ab113851; Abcam, Cambridge, MA, USA) was treated with an optimum concentration (20 μM) to make media colorless for 30 min, as per the manufacturer’s instructions. Then, the Hoechst (62,249, 1 µg/mL, Thermo Fisher Scientific, Marietta, OH, USA) was treated for 10 min to stain the nuclei.
4
Cellular ROS Quantification Assay
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C-33A and HeLa cells were treated with indicated concentrations. Cells were then washed two times with 1× buffer and stained by diluted DCFDA Solution (Abcam Plc., Cambridge, United Kingdom) for 45 min in an incubator. Then, the cells were washed two times with 1× buffer. The cells were observed using a fluorescent microscopy under low light conditions. Cellular ROS proportion were analyzed with Image J software and compared between the negative control (NC) and drug treated groups.
5
Measuring Oxidative Stress in viSMCs and viECs
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viSMCs and viECs were cultured with or without therapeutics for 7 days, then washed once with 1X buffer (Abcam). Cells were incubated with 10 µM DCFDA solution (Abcam) diluted in 1× buffer for 5 min at room temperature. After staining, cells were washed with 1× buffer twice and then imaged on a Zeiss 880 Airyscan confocal microscope at 20× magnification. Images were analyzed in FIJI for mean fluorescence intensity.
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