Nitric oxide assay kit

Caco-2 Cells, purchased from cell bank of the Chinese Academy of Sciences (Shanghai, China), were maintained in Dulbecco's modified eagle's medium (DMEM) with 20% fetal bovine serum (FBS, ThermoFisher, Cat. No. 10099-141) and 1% streptomycin/penicillin (ThermoFisher, Cat. No. 15140148). The Caco-2 cells were seeded into a 96-well microstate plate at a density of 2 × 10⁶ cells per well and allowed 24 h to adhere before introducing Lipopolysaccharides (1 μg/mL), and then co-incubated with or without increasing the concentrations of the isolates for 24 h at 37 °C. After treatment, the Nitric Oxide concentration in the medium was measured with Nitric Oxide Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) based on the nitrate reductase method. Absorbance was measured at 550 nm against a calibration curve with sodium nitrite standards. The nitric oxide concentration of the wells with cells was subtracted from the nitric oxide concentration of the blank wells without cells to calculate the nitric oxide production. Cell viability of the remaining cells was determined by an MTT (Sigma Chemical Co., St. Louis, MO, USA)-based colorimetric assay34 (link), 35 (link).

To test Ca²⁺, H₂O_2, ABA, PLA₂, NO, ABA and ET, adventitious roots (0.5 g, for NO and H₂O₂ assays, 0.1 g) were ground into homogenate on the rice with 4.5 mL PBS. The supernatant was collected at 4 °C for subsequent experiment after centrifugation at 3000 rpm for 20 min. Then the content of Ca²⁺, H₂O₂, NO was measured by Calcium Assay Kit, Hydrogen Peroxide assay kit (colorimetric method) and Nitric Oxide assay kit (Nitrate reductase method) (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China), and the content of ABA, PLA₂, ET were measured by Plant Abscisic Acid Elisa Kit, Plant PLA₂ Elisa Kit and Plant ET Elisa Kit (Senbejia Nanjing Biotechnology Co., Ltd, Nanjing, China) according the manufactures’ directions.