L 012

ROS were measured in eight-day-old seedlings. Seedlings were incubated in a 48-well microtiter plate containing 700 µL MGRL solution supplemented with 0.1% sucrose and 100 µM L-012 (a chemiluminescence probe; Wako, Japan). After 2 h incubation in 100 µM L-012 containing MGRL solution, 1 µM flg22 was added. A multi-label reader, VICTOR X3 (Perkin Elmer, USA), was used to verify the results we obtained from the L-012-derived chemiluminescence (CL; counts per second; cps) at 590-nm emission.

For in vitro measurements, neutrophils/macrophages were seeded on 96-well plates at a concentration of 10⁵ cells per well and infected with 10 MOI of ST-OVA. At indicated time points following infection, cells were loaded with 20 μM H₂DCFDA (Molecular Probes) in serum-free RPMI-1640 and incubated for 30 min at 37 °C. Cells were then washed twice with PBS, suspended in fresh RPMI-1640, no phenol red and readings were acquired at an excitation wavelength of 495 nm using a FilterMax F5 plate reader (Molecular Devices, CA, USA).
For ex vivo measurements, spleens were obtained from infected mice and single-cell suspensions were prepared. spleen cells (10⁶) were then loaded with 20 μM H₂DCFDA (Molecular Probes) in serum-free RPMI-1640 and incubated for 30 min at 37 °C. Cells were then suspended in fresh RPMI-1640, no phenol red and acquired immediately on CyAn ADP (Beckman Coulter). For in vivo ROS measurements, ST-OVA infected mice were anaesthetized with isoflurane, and injected (ip, 20 μg g⁻¹ of body weight) with L-012 (Wako, Osaka, Japan) which was dissolved in PBS. Bioluminescence signal was measured at 6 min post-injection of L-012 using an IVIS Spectrum imaging system (Perkin Elmer, MA, USA).