Dhr123

Dihydroethidium (DHE, Life Technologies) was used to evaluate production of reactive oxygen species (ROS) in mouse mesenteric arteries, as previously described [20 (link)]. Briefly, vessels were incubated with 5 µM of DHE for 20 min and subsequently observed under a fluorescence microscope (Zeiss Axio Observer A1—Obj. 20X). Images were acquired by a digital camera system (Olympus Soft Imaging Solutions). A second, estimation of total ROS production in mouse vessels was performed with the membrane-permeable fluorescent probe an analog of 2,7-dichlorodihydrofluorescein (DCDHF), dihydrorhodamine 123 (DHR123) (Invitrogen). After treatment, vessels were incubated with Krebs solution containing 5 μM DHR123 for 30 min at 37 °C, and then washed two times with PBS prior to fluorescence measurement using a fluorescence microplate reader (TECAN infinite 200 Pro).

Intracellular ROS levels were detected by staining with dihydrorhodamine 123 (DHR123, Sigma). Pseudozyma aphidis was grown in PDB for 2 days at 28 ^oC; cells were then spun down and discarded and the growth medium was filtered through a 0.4‐μm filter. Botrytis cinerea spore suspension (10⁵ spores/mL) was grown in a 48‐well plate containing PDB medium or PDB supplemented with P. aphidis secretions (50% and 75% of P. aphidis growth medium) or H₂O₂ (5 mm) for 14 h at 25 °C with agitation. Following incubation, DHR123 was added to a final concentration of 6 μm to each well and incubated for 1 h at 25 °C with agitation. We detected rhodamine fluorescence resulting from oxidized DHR123 by excitation at 485 nm, and measured the emitted light at 535 nm under a confocal microscope with argon ion and two He‐Ne lasers, or in a plate reader (Infinite F200, Tecan, Zurich, Switzerland). The fluorescence was recorded (four reads per well in triplicate per sample) and registered for every well (48‐well plate, 300 μL per well) every hour for 14 h. For microscopy, B. cinerea hyphae from each well were placed on a glass slide and excess PDB medium was removed with a pipette.

The blood leukocyte preparation was adjusted to 10⁶ cells/mL and placed in 96-well plates incubated with 0, 10, 25, 50 or 100 µg/mL Rooibos extract and dihydrorhodamine 123 (DHR 123, 1 μM, Sigma-Aldrich), and stimulated, or not (to check to the stimulation efficiency), by 1 µM PMA for 120 min, as previously described by Cholet et al. [28 (link)]. The fluorescence intensity of rhodamine 123, which is the product of DHR 123 oxidation by ROS, was recorded every 5 min for 120 min (excitation/emission: 485/538 nm) using a microplate fluorometric reader (Tecan Spark^®, Männedorf, Switzerland). Results were presented as a percentage of ROS production of stimulated treated cells compared to the control, which corresponded to stimulated untreated cells (100%).