Powershot g9

Cell viability was evaluated as mitochondrial metabolic activity [57 (link)] using the MTT assay as previously reported [10 (link)]. Briefly, the cells were plated onto 96-well plates (1 × 10⁴ cells/well), in DMEM with 10% FBS. After 24 h seeding, the cells were treated with different concentrations of AFPE or DMEM containing 0.2% PBS. After 24 h, 10 μL of the MTT solution (5 mg/mL) was added to each well in the dark, and the plates were incubated for 3 h at 37 °C under 5% CO₂ atmosphere. Following medium aspiration, double washing with 100 µl PBS, and solubilization of formazan crystals with 250 µL DMSO, the absorbance was measured at a wavelength of 570 nm using an ELISA plate reader (BioRad, Milano, Italy). The cell viability was expressed as a percentage relative to the untreated cells, cultured in a serum-free medium, set as 100%.
To evaluate cell morphology, the cells were seeded subconfluently onto a 24-multiwell, treated with different AFPE concentrations or in DMEM containing 0.2% PBS, and then observed for 24 h by a phase-contrast Zeiss Axiovert 40 CFL inverted microscope (Carl Zeiss, Milan, Italy) using a LD A-Plan 10×/0.50 P h 2 objective and equipped with a 12.1-megapixel CCD digital capture camera (Canon, PowerShot G9, Italy). Images were acquired using digital image software (Remote Capture DC, Canon).

Roots (0.5 g FW) were used for analysis of cell membrane damage (CMD) by measuring relative electrolytic leakage (EL), according to the method of Sullivan (1972 ). Cell death was detected by Evan's blue staining method (Gaff and Okong'O-Ogola, 1971 ). Samples were immersed in 0.1% Evan's blue solution for 30 min, and then washed with distilled water for 3 times to stop the color reaction. Staining reaction was photographed using a digital camera (PowerShot G9, Canon, Japan).

One day before experiments, L4 larvae were picked on NGM dishes (55 mm, 8 ml NGM) seeded with 320 µl OP50 culture. Spontaneous pumping of animals on food was video recorded (Powershot G9, Canon, Tokio, Japan) and visually counted for 20 s. Mean values, standard error of the mean (SEM) and further statistics (1-way ANOVA with Bonferroni post-hoc test) were calculated with OriginPro (OriginLab, Northampton, MA, USA).

The cell migration was evaluated by a wound assay [42 (link)]. Briefly, the cells (2 × 10⁵ cells/well) were seeded into a 6-well plate and incubated with complete medium at 37 °C and 5% CO₂. After 24 h of incubation, the cells were scraped horizontally and vertically with a sterilized P10 pipette tip (Gilson), subjected to different treatments in medium with 0.5% FBS, and two views on the cross of each well were photographed at 0, 12, and 24 h using a Zeiss Axiovert 40 CFL inverted microscope (Carl Zeiss, (MI), Italy), 10× objective. The microscope was equipped with a 12.1-megapixel CCD digital video camera (Canon, PowerShot G9, Italy) with a digital image software (Remote Capture Biomolecules 2019, 9, 833 5 of 17 DC, Canon, (MI), Italy). Quantitative analysis of the scratch assay was performed by measuring the gap area using the free image-processing software ImageJ, version 1.47 (https://imagej.nih.gov/ij/download.html, accessed on 18 November 2021).

Measurement of the solid-water contact angle is a direct method of assessing the degree of water repellency of a solid surface [30 ,31 (link)]. A homogeneous and absolutely flat surface is required to precisely measure the contact angle. Since it is impossible to obtain a sufficiently large flat area to measure a direct contact angle with a single soil particle, the sessile drop contact angle method (SDM) has been proposed [31 (link)].
One side of double-sided adhesive tape was attached to a slide glass, and then the oven-dried soil samples were sprinkled on the other side. The soil layer on the slide glass was compressed with a 100 g weight for 10 s, and then the slide was tapped carefully to remove unattached grains. After repeating this procedure twice, 10 drops of deionized water (2 ± 0.1 μL) were placed on the surface of the specimen with a micropipette (Axygen, AP-10, Corning, NY, USA) [31 (link)]. Horizontal images of the water droplets were captured within 5 s with a digital camera (Canon, PowerShot G9, Tokyo, Japan).

To determine the droplet size, an optical microscope (Zeiss Axiostar plus, Carl Zeiss AB, Stockholm, Sweden) with an integrated camera (Canon PowerShot G9, Canon, Solna, Sweden) was used. A small amount of CAP emulsion and placebo emulsion was placed between a coverslip and the glass slide. The pure API was suspended in isopropyl myristate and was also observed under a microscope directly, without the coverslip. Pictures at several fields were observed under polarized and non-polarized light.