Dp21 microscope

Migration assay was performed with transwell chambers (8 µm pore size, Corning, Australia). Cells grown as spheres and monolayer were enzymatically dissociated to single cells with Trypsin-EDTA treatment. Trypan Blue dye (Thermofisher, Australia) staining was performed to determine cell viability and cell count. 1 × 10⁵ of harvested cells from sphere or monolayer culture were plated into the upper chamber in a serum-free DMEM culture medium. A total of 500 µL of DMEM culture medium with 10% FBS was added into the lower chamber as the chemoattractant. Following 24 h of incubation at 37 °C, cells were treated with 4% paraformaldehyde (Fisher Scientific, Victoria, Australia) for 15 min for fixation. Next, cells were stained with 0.1% Crystal Violet (Sigma-Aldrich, New South Wales, Australia). The cells were removed from the upper chamber. Cells migrated onto the lower chamber were photographed under an inverted Olympus DP21 microscope equipped with a digital camera. For the quantification of cell migration, the Crystal Violet staining on the transwell membrane was extracted using 5% SDS and a 570 nm wavelength was used to measure absorbance [61 (link)].

Cells were seeded at density of 5 × 10⁴ per well into a 24-well plate. After cells reached confluency, a scratch or wound was made using a sterile pipette tip and washed with 1XPBS. Photographs were taken at the indicated time points with an inverted Olympus DP21 microscope (Olympus, Tokyo, Japan). The relative wound closure was quantified with the Fiji plug-in for Image J software version 1.53c (National Institutes of Health, Bethesda, MD, USA).

Roots were spread out in a 20 × 25 cm Perspex tray filled with 0.5 cm of and scanned using an Epson Perfection V700 photo dual-lens scanner with top lighting, with the following settings: 600 dpi, 8-bit grayscale, positive film. Obtained images were analyzed by the WinRhizo software (Version 2008a, Regent Instruments Inc) using a manual pixel classification with a gray-scale value of 225, to improve fine root detection. The debris filter was set to avoid counting debris with an area smaller than 1 cm², and a length/width ratio smaller than 5. The root system was divided into diameter classes of: 0–90, 90–250, 250–400, 400–600, 600–1,000 nm, which correspond to S-type laterals (<90 nm), L-type laterals (90–250 nm) and crown roots (250–1,000 nm). Details on branching frequencies or length of individual lateral roots were obtained using ImageJ as described by Wissuwa et al. (2020) (link). Micrographs obtained with an Olympus DP21 microscope (Olympus, Tokyo, Japan) with an Olympus UPlanApo lens at 4×, 10×, and 20× magnification were used to obtain exact measurements of root diameters.