Lumitrac 200

U87 cells (5000 cells/well) were seeded 24 h before experiment into 96-well plates. Before PBN incubation, the cell growth medium was replaced by 70 µL of fresh pre-warmed serum-free DMEM. Afterwards, 30 µL of the PBN solutions were added directly to the medium recovering the cells and incubated 1.5 h at 37 °C. Finally, 100 µL fresh DMEM supplemented with 20% FBS were added to the cells (10% FBS at final concentration) and the cells were incubated for further 36 h. The medium covering the cells was then carefully removed and 50 µL of 0.5 X Passive Lysis Buffer (PLB; Promega) were added for 30 min cell lysis at 4 °C. After a centrifugation step (10 min, 1,800 rpm, 4 °C), 10 µL of each cell lysate supernatant were transferred into a white 96-well plate (Greiner Lumitrac™ 200). Luciferase activity was quantified using a plate-reading luminometer (POLARstar Omega, BMG Labtech) and half-diluted dual luciferase assay reagents as described by the manufacturer (Promega). The results were expressed as percentage of relative light units (RLU) for each luciferase, normalized first to non-treated cells (%FLuc and %NLuc) and then to the value of %NLuc to obtain the relative Luc activity (%FLuc/%NLuc).

The plasmid was transferred into the A. tumefaciens GV3101 strain and injected into N. benthamiana leaves (grown in a greenhouse for two and a half weeks). After 48 h of injection, the injected N. benthamiana leaves were made into discs with a hole punch (diameter, 4 mm) and put into 96 empty plates (Lumitrac 200, Greiner, no.655075). The pieces were placed in each well individually, and 200 μL ddH₂O was added. Four replicates were taken for each sample. On the next day, water was replaced with a mock solution containing 34 mg l−1 (w/v) luminol (Sigma-Aldrich, Shanghai, China) and 20 mg l−1 (w/v) horseradish peroxidase (Sigma-Aldrich), and a ROS induced solution containing 34 mg l−1 (w/v) luminol (Sigma-Aldrich), 20 mg l−1 (w/v) horseradish peroxidase (Sigma-Aldrich) and 100 nM flg22. The luminescence was detected for 1 h with a signal integration time of 2 min using SpectraMax L microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Tomato plants (4-week-old) were exposed to either WL or WL+FR for 4 d. On Day 5, leaf discs (Ø 0.4 cm) originating from the third leaf of each plant were floated on deionized water for approximately 8 h in either WL or WL+FR corresponding to the light treatment conditions at room temperature without shaking. Each leaf disc was placed in each well of a white flat-bottomed 96-well plate (Greiner LUMITRAC 200) onto 180 µL of deionized water mixed with 20 µL of 10X reaction mix (200-µM Luminol L-012 and 10-µg mL⁻¹ horseradish peroxidase) with a final concentration of 20-µM Luminol and 1 µg mL⁻¹ of peroxidase. The luminescence was quantified by using a GloMax luminometer (Promega). The background noise was measured for 15 min prior to adding 1-µM flg22 or 1% chitosan (w/v) (final concentrations) on the leaf discs. The luminescence was measured for ∼1 h by recording 34 cycles of 100 s (Albert and Fürst, 2017 (link)).

Apoplastic ROS were measured using GloMax-Multi + Detection system (Promega, United States) using a protocol modified from an earlier described protocol (Bisceglia et al., 2015 (link)). Briefly, biopsy punches (4 mm diameter, Kai Medical, Japan) were used to collect four leaf disks per plant from the mesophyll of the second true leaf. These samples were collected from six 4-week-old tomato plants that had not been previously sprayed. The collected leaf disks were placed in a Petri dish containing distilled water for 2 h in the dark. Every 30 min, the distilled water was replaced to remove cellular damage-related compounds. Leaf disks were placed in 96-well microplates (Lumitrac 200, Greiner Bio-One, United States) with 100 μl MS medium (4.43 g/l MS pH 5.7, Duchefa Biochemie, Netherlands) and 50 μl luminol/peroxidase solution containing 9 μg/ml luminol and 4 μg/ml peroxidase (Sigma-Aldrich, United States). After 4 h incubation at 24°C, 50 μl of 62.5 mg/l COS-OGA for elicited leaf disks or 50 μl 0.1% Tween 20 for control leaf disks were added. The chemiluminescence was measured directly for 90 min with a signal integration time of 1 s. Each curve was integrated and expressed in relative chemiluminescence units. The average value of chemiluminescence per treatment was expressed in percentage of the control (n = 12).