Dcfh da assay

The fluorescent dichlorofluorescin diacetate (DCFH-DA) assay (abcam, Cambridge, UK) was used according to the manufacture’s 24–48 h treatment protocol to analyze the production of reactive oxygen species in cells exposed to Doxorubicin or tert-butyl H₂O₂ (TBHP). Autobioluminescent T47D or HepG2 cells were seeded at ~2.5 × 10⁴ cells/well in flat-bottom black 96-well plates and incubated under standard conditions. After overnight incubation and prior to compound treatment, attached cells were washed with 100 μL 1 × phosphate buffered saline (PBS) and then treated with 100 μL of the test compound at concentrations ranging from 1 nM to 10 µM, or with 0.1% DMSO as a control. Each compound was tested in triplicate plates and each concentration was tested in triplicate wells per plate. After treatment for 23 h, 100 μL of 60 μM 2′,7′-dichlorofluorescin diacetate (DCFH-DA) was added to each well and the cells were incubated for an additional 30–60 min. After 24 h of total incubation, fluorescence was measured using a 485 nm excitation and a 535 nm emission wavelength in the CLARIOstar plate reader (BMG Labtech Ortenberg, Germany).

For oxidative stress generation, 500 µM of TBH (tert-butyl hydroperoxide, Luperox^® TBH70X, Merck Life Science S.r.l., Italy) was used for 2 h, alone and in combination with grapefruit-seed-extract treatment. The control (Ctrl) groups received an equal volume of the medium. For the DCFH-DA assay (Abcam, Milan, Italy) extracts were utilized at 100 µg/mL for 2 h. The treated and control cells were analyzed by using microscopy (Axio Scope 2 microscope, Zeiss, Germany). At the end of the treatments, each sample was added to DCFH-DA (100 µM) and placed in the dark for 10 min at room temperature. After washing with PBS, cells were analyzed using a Microplate Reader GloMax fluorimeter (Promega Corporation, Milan, Italy) at the excitation wavelength of 475 nm and emission wavelength 530 nm for fluorescence intensity detection, and results were expressed as a percentage of the control group. Cell fluorescence was also visualized using the fluorescence microscope Zeiss Axio Scope 2 microscope (Carl Zeiss, Oberkochen, Germany).

The levels of intracellular reactive oxygen species (ROS) were measured using the dichlorodihydrofluorescein diacetate (DCFH-DA) assay (Abcam) according to the manufacturer’s guidelines. DCFH-DA is a lipophilic cell permeable compound that is deacetylated in the cytoplasm by cellular esterases, and later oxidized by ROS to a highly fluorescent molecule. THP-1 monocytes were differentiated into macrophages and loaded with 20 μM DCFH-DA in PBS for 30 min at 37 °C. Thereafter, cells were treated with the NFC suspensions (50, 100, 250 500 μg/mL) and fluorescence was recorded every 30 min over 120 min (excitation 485 nm, emission 535 nm) at 37 °C using a plate reader (Tecan Infinite M200). Tert-butyl hydroperoxide (TBHP, 50 μM) was used as positive control.