Model infinite 200 pro

The determination of intracellular oxidant stress was based on the oxidation of DCFH-DA. Briefly, CAL-27 cells were seeded in 60 mm dishes and treated with NRC-03 in the absence or presence of other test compounds for the indicated time, and then were incubated with redox-sensitive dye DCFH-DA at 37 °C for 30 min and analyzed by a flow cytometer.
For determining mitochondrial ROS production, CAL-27 cells were cultured in confocal dishes and treated with or without other test compounds for 1 h prior to NRC-03 for 4 h. The cells were stained with 100 nM Mitotracker green for 30 min, and then with 2 μM MitoSOX Red (excitation: 510 nm, emission: 580 nm) for 15 min at 37 °C and visualized with CLSM. The ratio of fluorescence intensities was determined by ImageJ software.
For determining hydrogen peroxide (H₂O₂) production, CAL-27 cells were cultured in a 96-well plate (8 × 10⁴ cells/well) with a clear bottom and black sides for 24 h. The cells were treated with or without other test compounds for 1 h prior to NRC-03 treatment for 4 h. The quantification of H₂O₂ production was assessed by AbIR Peroxidase Indicator using a Hydrogen peroxide assay kit – (Fluorometric-Near Infrared) complemented with a fluorescence plate reader (Model Infinite 200 Pro, Tecan) at room temperature (Ex 640/Em 680).

The oxygen consumption rate (OCR) was evaluated using an oxygen consumption rate assay kit. Briefly, CAL-27 cells were cultured in a 96-well plate (8 × 10⁴ cells/well) with a clear bottom and black sides for 24 h. Next, 100 μl of medium mixed with different concentrations of NRC-03 and 5 μl of oxygen fluorescent probe was added to each well. Thereafter, blocking buffer (2 drops/well) was immediately added to each well to prevent external oxygen generation. After that, the plate was read with a fluorescent microplate reader (Model Infinite 200 Pro, Tecan) at 37 °C (1 read per 3 min, Ex 455/Em 603). Since the fluorescence of this oxygen probe can be quenched by O₂, the value of the fluorescence signal was inversely proportional to the amount of O₂ in each well. OCR was calculated based on the changes of fluorescence signal over 2 h as follows: OCR (%) = (final fluorescence in NRC-03-treated cells−initial fluorescence in NRC-03-treated cells)/(final fluorescence in control cells−initial fluorescence in control cells) × 100%.

A whole-cell enzyme-linked immunosorbent assay was performed using monoclonal antibodies 6E4 and 3F11 by the method of Abdillahi and Poolman (37 (link)). Bacteria were grown on sRPMI plates, which were supplemented with Neu5Ac where indicated. Cells were harvested and resuspended in phosphate-buffered saline (PBS) to an optical density at 600 nm of 0.10. Microtiter plate wells were filled with 100 µl of the bacterial suspension and dried at 40°C overnight. The wells were washed, and ELISA was performed with MAb 6E4 at a dilution of 1:100. Secondary antibody (alkaline phosphatase-conjugated goat anti-mouse IgG; Jackson ImmunoResearch, West Grove, PA) was used at a dilution of 1:10,000. The substrate p-nitrophenylphosphate was applied at a concentration of 1.0 mg/ml. Absorbance was read in a Tecan Model Infinite 200 Pro plate reader at 405 nm.