Measuring Intracellular ROS Using DCFH-DA

Intracellular ROS was measured using 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA), a membrane-permeable dye that becomes fluorescent when oxidized by H₂O₂ and widely used to study the ROS scavenging capability of nanoceria [44 (link), 45 (link)]. Cells (10⁵ cells/well in 1 ml of medium) were seeded into 12-well plates and incubated for 16 h at 37°C before the addition of the nanoparticles (1.5 μg/ml; 15 pg/cell) or equivalent amounts of heparin (0.041 μg/ml Hep, 0.011 μg/ml L-hep-A or 0.054 μg/ml L-hep-B). Cells were harvested after 24 or 72 h using TrypLE™ Express Enzyme (1×) (ThermoFisher Scientific). Cells were resuspended in flow buffer (2 mM EDTA, 0.5 w/v% bovine serum albumin (BSA) in PBS) and 10⁵ cells were resuspended in DCFH-DA (0.5 μM in flow buffer) and incubated for 20 min at 37°C before being washed with and resuspended in flow buffer (200 μl). Propidium iodide (PI; 2 μg) was added to each tube and mixed for 5 min before samples were analysed in a flow cytometer (LSRFortessa™ SORP, BD, Franklin Lakes, NJ) for fluorescence intensity (DCF and PI), forward scatter and side scatter (SSC) for 10⁴ cells after gating for live cells. Data were analysed with the FlowJo_V10 software (BD).