Fluorescence microplate reader

The amount of liposomally encapsulated SLP in each formulation was quantified via the fluorescent signal of the NBD group (λ _excitation = 462 nm, λ _emission = 540 nm). In order to calculate the loading efficiency and total drug loading of the SLPs the liposomal samples and filtrates (containing the non-encapsulated SLP) were collected during the purification/concentration steps. Calibration curves were prepared for all SLPs. In order to exclude possible interference due to the presence of lipids a series of empty liposomes spiked calibration curves were prepared as well. All measurements were performed in 1:1 (v/v) MeOH/PB and data was acquired with a fluorescence micro plate reader (Tecan, Salzburg, Austria). For every formulation the loading efficiency (Eq. 1) and peptide recovery were calculated (Eq. 2). $$\mathit{\text{Loading efficiency}}\left(\%\right)=\left(\frac{\mathit{\text{Total peptide}}-\mathit{\text{free peptide}}}{\mathit{\text{Total peptide}}}\right)x100\%$$
In equitation 1 the total peptide accounts for the total amount of SLP in the liposomal dispersion prior to purification. The free peptide is the amount non-encapsulated SLP that was determined after purification. $$\mathit{\text{Peptide recovery}}\left(\%\right)=\left(\frac{\mathit{\text{Encapsulated peptide}}}{\mathit{\text{Total added peptide}}}\right)x100\%$$
In equitation 2 the encapsulated peptide accounts for the amount of SLP in the purified and concentrated liposomal dispersion. The total added peptide accounts for the amount of SLP that was added during liposome formulation.

The CellTiter-Lumi^TM Plus Luminescent cell viability assay kit (Beyotime, Shanghai, China) was used to determine the cell viability following the manufacturer’s protocol. Briefly, GCs were seeded in a 96-well plate at a density of 1 × 10⁵ cells/well, and incubated overnight at 37 °C in humidified air containing 5% CO₂. The cells were equilibrated for 10 min at room temperature. Then, 100 μL of the CellTiter-Lumi^TM Plus detection reagent was added to each well, and vibrated for 2 min at room temperature to lyse the samples. After an additional 10 min incubation at room temperature, the luminescent signals were measured by a fluorescence microplate reader (Tecan, Shanghai, China).

The relative levels of intracellular ROS were measured using a commercial ROS detection kit (Beyotime, China). Briefly, IPEC-J2 cells were cultured in 6-well plates for 16 h before treatment, pretreated with RSV (0 μM, 25 μM, and 50 μM) for 4 hours, and then exposed with H₂O₂ (500 μM) for 4 h to induce ROS production. Cells were washed twice with PBS and then incubated with 5 μM 2′,7′-dichlorofluorescein diacetate (DCFHDA) for 30 min. Fluorescence intensity was measured using a fluorescence microplate reader (Tecan, Sunrise) at excitation/emission wavelengths of 525/610 nm.

Treated cells were added DCFH-DA diluted in serum-free medium at a ratio of 1:1,000, Incubate at 37°C for 20 min, Observing the fluorescence expression of ROS under fluorescence microscope (Leica, Germany). Fluorescence intensity was measured by fluorescence microplate reader (TECAN, Switzerland) at 485 nm excitation and 525 nm emission.

The Quant-iT™ PicoGreen dsDNA Assay kit was used to quantify the level of NETs according to the manufacturer’s instructions. Briefly, PMN from each lactational stage group were diluted with RPMI 1640 medium, and 200 μL (5 × 10⁵ cells) were inoculated into 96-well plates (in triplicate) and cultured for 3 h at 37 °C and 5% CO₂. At the end of the culture, a pipette was used to blow each well to break up the long dsDNA. Samples were then centrifuged at 500× g for 5 min, and 100 μL supernatant was transferred to a new black 96-well plate, and 100 μL Quant-iT™ PicoGreen working fluid to each well was added, gently mixed, and incubated in the dark at room temperature for 3 min. The fluorescence intensity of the samples was measured on a TECAN fluorescence microplate reader (Bern, Switzerland) at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. The fluorescence intensity of each sample was calculated relative to the fluorescence intensity of blank samples (no cells).

The whole body H₂O₂ concentration in pea aphid was determined as described previously [19 (link) and 67 (link)]. The pea aphids were collected in 50 mM sodium phosphate buffer (pH 7.4) containing 2 mg/mL catalase inhibitor 3-amino-1, 2, 4-trizole (Sigma, St. Louis, MO, USA). After homogenization, the samples were filtered through a 10-kDa molecular weight cutoff spin filter (Millipore, Billeeica, MA, USA). The eluent was collected and the H₂O₂ concentration was measured with a Hydrogen Peroxide Assay Kit (Invitrogen, Carlsbad, CA, USA) on a fluorescence microplate reader (Tecan, Männedorf, Switzerland) according to the manufacturer’s protocol. The values were normalized to the total amount of protein in the samples. Ten pea aphids from each group were evaluated in the assays.