A live cell, high-throughput, microplate-based ROS assay that utilized the cell permeable substrate, 2',7'-dichlorofluorescin diacetate, (DCF-DA) a reliable fluorogenic marker for ROS detection was used as previously described [10] . Upon ROS generation, the highly fluorescent dye, 2',7'-dichlorofluorescein is produced, with EX (excitation): 495 nm and EM (emission): 530 nm. In brief, DLD-1.ApoL6 cells were plated in 96-well tissue culture plates at a density of 100,000 cells/well and then loaded with DCF-DA at 20 μM final concentration for 30 min before treating with the indicated compounds at 2, 10 and 50 μM in D.0 for 8 hrs. Both end point and area-scan readings were taken using the Synergy H4 Plate Reader at 485 nm with emission at 528 nm using a 20 nm bandwidth, according to the manufacturer's instruction.
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Microplate-based ROS Assay with DCF-DA
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Other organizations : University of New Mexico, Wuhan Polytechnic University, Hunan Normal University, Institute of Subtropical Agriculture, Penn State Milton S. Hershey Medical Center, Florida International University
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Variable analysis
independent variables
- Concentrations of the indicated compounds (2, 10, and 50 μM)
- Reactive oxygen species (ROS) levels measured by the fluorescence of 2',7'-dichlorofluorescein (EX: 495 nm, EM: 530 nm)
- Cell line: DLD-1.ApoL6 cells
- Cell density: 100,000 cells/well
- Dye: DCF-DA at 20 μM final concentration
- Incubation time: 30 min for dye loading, 8 hrs for compound treatment
- Plate reader settings: Synergy H4 Plate Reader, excitation at 485 nm, emission at 528 nm with 20 nm bandwidth
- No positive or negative controls were explicitly mentioned in the protocol.
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Plating cell density: 100,000 cells/well (Input protocol), 7,000 cells/well (Protocol 1), 1 x 10^4 cells/well (Protocol 2), 'Cells were seeded in 12-well plates' (Protocol 3), no specific density mentioned (Protocols 4 and 5). Optimized cell density is important for consistent ROS measurements.
Incubation time with DCF-DA: 30 min (Input protocol), 45 min (Protocols 1 and 2), 25 min (Protocol 3), no specific time mentioned (Protocols 4 and 5). Standardizing the DCF-DA incubation time is crucial for accurate ROS detection.
Excitation and emission wavelengths: Ex 495 nm, Em 530 nm (Input protocol), Ex 485 nm, Em 520 nm (Protocol 1), Ex/Em 485/528 nm (Protocol 2), Ex 488 nm, Em 525 nm (Protocol 3), Ex 485 nm, Em 535 nm (Protocol 4), Ex 492 nm, Em 515 nm (Protocol 5). Ensuring consistent excitation and emission wavelengths is critical for reproducibility.
Positive control: H2O2 100 μM (Protocol 1), H2O2 1 mM (Protocol 2). Using a positive control, such as H2O2, helps to validate the assay performance and ensure the cells are responding as expected.
Blank control: 'Blank wells (no cells) were also used as a control' (Protocol 2). Including a blank control without cells is important to account for any background fluorescence or interference.
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