Multiskan microplate photometer

The stable conversion product of NO, nitrite (NO₂⁻), was measured to assess the NO concentrations in cultures. An aliquot of culture supernatant was added to an equivalent volume of Griess reagent (a mixture of 1% sulfanilamide plus 0.1% N-(1-Naphthyl) ethylenediamine dihydrochloride in 5% H₃PO₄) in 96-well plates and incubated for 10 min at room temperature. Next, nitrite levels were measured colorimetrically using a Multiskan microplate photometer (Thermo Fisher Scientific) at 540 nm. Finally, the concentration value was calculated by comparing to the standard curve made by sodium nitrite.

For GN6 ELAA using bacterial OMVs, Nunc-Immuno 96 MicroWell solid plates (Thermo Scientific, USA) were used to immobilize bacterial OMVs in Tris buffer. After incubating OMVs at various concentrations in the plate overnight at 4 °C, the plate was washed twice and blocked using 2% BSA-Tris buffer for 2 h. After blocking, 20 pmol of GN6 aptamer and N40 control were separately added and incubated for 1 h. After washing 4 times, streptavidin-Poly HRP conjugate (Pierce, USA) was added and incubated for 30 min. After thoroughly washing 5 times in Tris buffer with 0.05% Tween-20, Ultra TMB-ELISA reagent (Thermo Scientific, USA) was added. After 15 min, 1 M sulfuric acid as stop solution was added. Absorbance at 450 nm was measured using Multiskan microplate photometer (Thermo Scientific, USA). The measured values were analyzed using non-linear regression fit model of SigmaPlot 12.0.

After aspirating the water on the surface of the duckweed, 0.1 g of the sample was weighed in a 10 mL centrifuge tube, placed in a −20 °C freezer for 1 h, removed to add 2 mL of 95% ethanol, preheated to 50 °C, shaken thoroughly, and placed in the dark at room temperature for 3 h. The supernatants were then collected. Absorbances of the chlorophyll pigments in the collected supernatants were measured at 663 and 645 nm using a full-wavelength Multiskan microplate photometer (Thermo Scientific Multiskan FC, Shanghai, China), and the chlorophyll content was calculated according to Equations (2)–(4).



where Chl a is the concentration of chlorophyll a (mg/L), Chl b is the concentration of chlorophyll b (mg/L), Chl is the concentration of chlorophyll (mg/L), A₆₆₃ is the absorbance of the chlorophyll solution at 663 nm, and A₆₄₅ is the absorbance of the chlorophyll solution at 645 nm converted to chlorophyll content per gram of fresh leaves (mg/g) based on the chlorophyll concentration in the extracts.

Neurotoxicity was assessed by determining N2a cell viability. An MTT reduction assay was used to measure the metabolic activity of living cells and reveal the data proportional to the number of viable cultured cells. MTT (0.5 mg/mL) dissolved in phosphate buffered saline (PBS) was added to the cultures for 2 h of incubation at 37 °C. After removing the medium containing MTT, the purple formazan crystals formed were dissolved with dimethyl sulfoxide. The absorbance of the resulting solution was obtained by measuring at 570 nm using a Multiskan microplate photometer (Thermo Fisher Scientific, Waltham, MA, USA) and subtracting the reading at 620 nm (reference wavelength). Blank values were subtracted from each reading, and the absorbance was expressed as a percentage relative to the corresponding control.