Black 96 well plate

For fluorescence measurement, EVs were recovered as described in Section 4.3, then resuspended in the same amount of PBS and transferred into a 96-well black plate (Greiner, Frickenhausen, Germany). Fluorescence was measured on an Infinite^® 200 PRO microplate reader (TECAN, Männedorf, Switzerland), at excitation and emission wavelengths of 580 and 620 nm, respectively.

In vitro YLG hydrolysis assays were performed using 3 μM of ShHTL7, ShHTL7‐L143Y, AtD14, and OsD14 in a reaction buffer (1× PBS) with 0.1% dimethyl sulfoxide (DMSO) in a 100 μl volume on a 96‐well black plate (Greiner). The fluorescent intensity was measured by SpectraMax i3 (Molecular Devices) using an excitation wavelength of 480 nm and a detection wavelength of 520 nm. ShHTL7, ShHTL7‐L143Y, AtD14, and OsD14 were mixed with Triton X‐100, using serial dilutions to cover the Triton X‐100 concentration range from 5 μM until 4.87 nM. Protein‐Triton mixes were pre‐incubated for 30 min. 1 μM YLG (Tokyo Chemical Industry Co. Ltd.) was then added, and the assay was incubated for 60 min. The change in fluorescence observed over the course of 1 h incubation of YLG in buffer without protein was subtracted from the data collected in the presence of protein. The resulting relative fluorescence values were analyzed. The inhibitory curves and IC₅₀ values were plotted using GraphPad (Prism 6) four‐parameter logistic curve. The rate of YLG hydrolysis by ShHTL7 was recorded following the above method at 10‐min interval for 2 h.

Absorbance spectra were measured using a UV-Visible CLARIOstar plate reader (BMG Labtech) in a UV-Star 96-well plate (Greiner). Fluorescence spectra with visible excitation were collected using the same instrument in epifluorescence mode using a 96-well black plate (Greiner). Emission spectra with DUV excitation were collected on a PC1 photon-counting spectrofluorometer (ISS), which uses xenon arc lamp excitation.
Samples were placed in quartz cuvettes (Starna) for the measurements. Cuvettes were cleaned using a Kontes vacuum cuvette washer (Kimble) connected to house vacuum. They were first rinsed with dilute soapy water, then distilled water, then ethanol, and then finished with distilled water. If contamination was detected in blank runs with ultrapure water, cuvettes were cleaned with sulfuric acid (H₂SO₄) before vacu-washing.
Quantum yields were derived by obtaining the slope of the curve of absorbance A versus integrated emission I and using the formula
$$QY=Q{Y}_{ref}\left(\frac{I}{A}\right)\left(\frac{A_{ref}}{I_{ref}}\right),$$
where “ref” refers to the reference fluorophore, which here was fluorescein (for 480 nm excitation) or tryptophan (for 275 nm excitation) or quinine (for 350 nm excitation). All solutions were in water or buffered aqueous solutions with refractive index 1.33, and at least six dilutions were measured for each sample to obtain the linear range of the absorbance curves.

Discs from young leaves of Ec1-, SlEpk1- and NbFls2-silenced plants were excised with a 4-mm-diameter cork borer. Leaf disks were floated adaxial side up in a 96-well black plate (Greiner Bio-one, Kremsmuenster, Austria) containing 180 μl of water per well, and left at room temperature overnight. The next day, the water was removed, and 100 μl of a solution containing the following was added: 500 nM flg22 (GenScript, Piscataway, NJ USA), luminol at 34 μg/ml, and horseradish peroxidase at 20 μg/ml (Sigma, St Louis, MO, USA) in water. Luminescence was measured using the Synergy HT plate reader (Biotek, Winooski, VT, USA). Four leaf disks per plant were taken, and six plants silenced for each gene were considered in each experiment.

In the second trial, 0.6 mg/g of fluorescein isothiocyanate-dextran 4 (FITC-dex 4; Sigma-Aldrich) dissolved in PBS was administered intragastrically to each mouse. Blood samples were collected after 3.5 h as described above, and 80 μl of serum was transferred to a 96-well black plate (Greiner). The concentration of FITC-dex 4 was determined using fluorescence spectrophotometry (excitation: 488 nm; emission: 520 nm; Infinite M200, Tecan); serially diluted FITC-dextran was the standard (range: 0–12,000 μg/ml).

Intracellular ROS formation was detected using 2′,7′-dihydrofluorescein diacetate (DCFH-DA, Sigma chemicals Co., St. Louis, MO, USA). A nonfluorogenic compound DCFH-DA is oxidized to fluorescent compounds DCF by hydroxyl radical, peroxyl radical, and other reactive oxygen species (ROS) within the cell [15 (link)]. HaCaT cells were seeded at the density of 2 × 10⁴ in a 96-well black plate (Greiner Bio-one GmbH, Frickenhausen, Germany) for microplate reader and a chamber slide (ThermoFisher, Waltham, MA, USA) for fluorescence microscope.
After MSAE treatment for 18 h and 1.6 mM H₂O₂ treatment for 30 min, the cells were incubated with 25 μM DCF-DA in culture medium at 37°C for 30 min in the dark. Intracellular ROS as indicated by DCF fluorescence was measured using an EVOS fluorescence microscope (ThermoFisher, Waltham, MA, USA) and an Infinite M200 microplate reader (Tecan, Männedorf, Switzerland) with excitation and emission wavelengths of 485 nm and 530 nm, respectively.