Perfection v800

Cells were plated on 96-well plates (2000 per well) with a volume of 100 µL cell culture medium on day 0. MABS treatment was performed on day 1. On day 7, plates were fixed for 10 min with 96% methanol, stained with 0.1% crystal violet, and washed with dH₂O. Dried plates were scanned (Epson Perfection V800, Epson, Suwa, Japan; Settings: Positive film mode, 600 dpi, saved as TIF-format) and analyzed with ImageJ software (NIH, Bethesda, MD, USA). Cell survival graph preparation and EC₅₀ calculations were achieved with GraphPad Prism software (La Jolla, CA, USA). For RONS scavenger experiments, N-acetyl-L-cysteine (NAC) (Sigma Aldrich, St. Louis, MO, USA, CAS no. 616-91-1) diluted in dH₂O was added to the media prior to MABS treatment.

EBT3 GafChromic films (Ashland, Parlin, New Jersey) from a single batch were used for all measurements. Calibration films were irradiated based on exposure time from the corresponding ion chamber measurements for the in-air calibration protocol. We waited at least 20 h before scanning each film. The film was contained first in envelopes and then within a light tight container. An Epson Perfection V800 (Seiko Epson Corporation, Suwa, Japan) was used for film scanning. A calibration curve was generated for the batch of film. The films were scanned in 48-bit mode at 75 dpi in transmission mode, with no image corrections applied. The calibration curve was generated using SNCPatient software (SunNuclear, Melbourne, Florida), and all films were imported into the program. Analysis was completed using an in-house Python script. For each petri dish, a central square region of interest (ROI) was calculated based on the size and resolution of the scanning mode. The central ROI consisted of 50% of the nominal diameter of each petri dish well, unless noted otherwise. The mean dose of each film was reported and compared with the calculated D_{w,z = 0} (the calibrated dose output at the surface of a water phantom as defined by TG-61 using the in-air method) delivered.

For clonogenic survival assay, 4 × 10⁵ asynchronously growing RPE-1 cells were counted and plated in 6 cm dishes. The day after plating, cells were treated with the indicated doses of X-rays. Soon after irradiation, cells were counted and plated in triplicates in 6-well plates, at a density of 250 cells/well. Colonies were grown for 14 days at standard cell culture conditions with medium change every two days. At the assay endpoint, colonies were fixed with 3.7% formaldehyde (Chemie Brunschwig, extra-pure 37-41% solution diluted 1/10 in deionized water) for 10 min before staining with 0.5% Crystal Violet in 10% Ethanol solution (Honeywell) for 10 min at RT. Last, staining solution was discarded, colonies were washed 3 × 2 min in PBS while shaking and left to dry overnight. Images of the plates were acquired using a flatbed scanner Epson perfection V800 (Epson, Nagano – Japan) with the following settings: 24-bit colour, 1200 dpi resolution. Quantification was performed using the ColonyArea plug-in of Fiji^{40 (link)}.

Plant height, leaf lengths and number of leaves were manually measured throughout the study. Root phenotype was scanned using an optical scanner (Epson Perfection V800, Japan) and analyzed using the WinRHIZO Arabidopsis 2019 program (Regent Instruments, Canada). Root length and surface area were calculated using the method previously described (Suwanchaikasem et al., 2022 (link)). Shoot and root fresh weights (FWs) were measured on the collection day using an analytical balance (Ohaus, USA).

For the spot assay, cultures were grown to log phase in a liquid medium and serial fivefold dilutions of cells were spotted on plates and incubated at 30°C. After colony formation, the plates were scanned using an Epson Perfection V800 photo scanner.

Nine-day-old in vitro grown A. thaliana seedlings were individually inoculated with increasing densities (P_i) of surface-sterilized J2s of H. schachtii (0–50 juveniles ml^–1 of modified KNOP medium). Roots of nematode-infected plants were scanned at 7 days post-inoculation (dpi) using an Epson Perfection V800 photo scanner. Various root measurements were conducted (i.e. total root length, main root length, total secondary root length, and average secondary root length), collectively referred to as the root system architecture. Measurements were taken using the WinRHIZO package for Arabidopsis (WinRHIZO pro2015, Regent Instrument Inc., Quebec, Canada). The number of root tips was counted manually based on the scans made by WinRHIZO. Differences in the root length per seedling in centimetres and the number of root tips were statistically analysed with a one-way ANOVA with post-hoc Tukey’s HSD test in R.