Selenite

Volumes specified for 35 mm well (1 well of a 6-well plate).
On day 0 of iPS-microglia differentiation, plate HPCs at 10,000 cells per cm² onto 1 mg/mL Matrigel-coated plates (100,000 per 35 mm well). Plate cells into iPS-microglia medium at 2 mL per 35 mm well: DMEM/F12, 2X insulin-transferrin-selenite, 2X B27, 0.5X N2, 1X glutamax, 1X non-essential amino acids, 400 μM monothioglycerol, 5 μg/mL insulin. Immediately before use, microglial medium should be supplemented with 100 ng/mL IL-34, 50 ng/mL TGFβ1, and 25 ng/mL M-CSF (Peprotech) taken from single-use frozen aliquots (important: do not freeze/thaw these cytokines as it will significantly impair differentiation and yield as well as induce activation. It is crucial to thaw cytokines immediately before use). Throughout the differentiation of HPCs to microglia, these cells will predominantly grow non-adherently. On days 2, 4, 6, 8, and 10, add 1 mL fresh media plus freshly thawed tri-cytokine cocktail. Cytokines are diluted to the concentrations listed above before adding to conditioned medium. Do not fully remove media during the microglial differentiation as the cells secrete paracrine cytokine signals and will not properly differentiate upon removal of those. On day 12, collect 6 mL media from each 35 mm well leaving 1 mL conditioned medium on the plate. Centrifuge non-adherent cells in removed medium for 5 min at 300 x G. Aspirate medium and resuspend non-adherent cells in 1 mL fresh medium plus tri-cytokine cocktail per 35 mm well and add back to the same well which contains the 1 mL conditioned medium. Continue to supplement media (1 mL) on days 14, 16, 18, 20, 22, and 24. On day 25, centrifuge cells leaving 1 mL conditioned media per 35 mm well as on day 12. On day 25, cells should be resuspended in microglia media plus 100 ng/mL IL-34, 50 ng/mL TGFβ1, 25 ng/mL M-CSF, 100 ng/mL CD200 and 100 ng/mL CX3CL1 to further mature microglia and ensure homeostasis. On day 27, feed cells with microglia media with five cytokine cocktail (1 mL per well). On day 28 cells collected for RNA sequencing or use for transplantation or functional assays. If necessary, cells can be maintained for 1–2 additional weeks via media supplementation as above, although longer-term culture is not advised.

Trees were sprayed with I, Se and Ca, 2 weeks before harvest—BBCH 81 (on 12 and 15 September 2020 and 2021, respectively). The tested spray solutions contained I as K-iodide (KI; 76.5% I) or K-iodate (KIO₃; 59.3% I), Se as Na-selenite (Na₂SeO₃; 45.7% Se) or Na- selenate (Na₂SeO₄; 41.8% Se), and Ca as CaCl₂ (28% Ca) at amounts of 0.5 kg I, 0.25 kg Se and 7 kg Ca per ha. The salts of I and Se were technical grade chemicals (Sigma-Aldrich, Poznań, Poland), whereas CaCl₂ was applied as a commercial fertiliser (CIECH, Warsaw, Poland). The rates of I, Se and Ca used in the spray treatments were high, considering those applied in apple orchards by Budke et al. [15 (link)] and Wójcik and Wójcik [21 ] for I, by Groth et al. [19 (link)] and Budke et al. [15 (link)] for Se, and by Wójcik [27 ] and Raese and Drake [28 ] for Ca.
To examine the effects of species of I and Se used in the spray solutions containing CaCl₂ on the uptake rate of exogenously applied Ca by apples, we tested also CaCl₂ spray performed at the same timing and dose as the combined spray treatments of I, Se and Ca. Control trees were not sprayed with either I, Se and Ca or water. The acronyms of the tested spray combinations and the solution concentrations used are given in Table 2.
To improve wetting of fruit surfaces by the tested spray solutions, the non-ionic surfactant Tween^® 20 (polyoxyethylene sorbitan monolaurate; Sigma-Aldrich, St. Louis, Missouri, USA) was added at a rate of 0.3 L ha^-1. The sprays were applied with a cross-flow orchard sprayer with an air deflector (Agrola Turbo 1000v Optimum; AGROLA, Platkowice, Poland) equipped with hollow-cone nozzles using 750 L of water per ha. The spray treatments were performed on both sides of the tree rows in the morning (09:00–11:00 h) when leaves/fruit were dry, the wind velocity was < 2 m s^-1, air temperature varied from 10 to 14°C, and the relative air humidity (RH) was within the range of 75–89%.
Over the entire period of the study, the same trees were used for the given spray combinations. The experiment was set up in a completely randomised block design with four replications, with a replicate of each combination within one row. Each plot consisted of 10 trees. Plots within a row were separated by two trees. A buffer row was left untreated between every two adjacent sprayed rows to reduce contamination between treatments.

SKOV-3 and OVCAR-3 cells were cultured in growth medium to 80%confluency on sterile plastic petri dishes (Corning, UK) at 37 °C and 5% CO2. After 48 h incubation culture medium was removed and 2 mL of fresh medium containing aqueous Se (Na₂SeO₃) or SeNPs. SKOV-3 cells were treated with IC20; selenite 3 μg/mL, BSA-SeNP 6 μg/mL or chitosan-SeNP 13 μg/mL. OVCAR-3 cells were treated with selenite 40 μg/mL, BSA-SeNP 20 μg/mL or chitosan-SeNP 18 μg/mL. Following addition of selenium treatment cells were incubated for a further 48 h prior to analysis. A minimum of three biological repeats were conducted for each cell type and treatment.

Cells were treated for 24 h with SeNP or selenite with (mentioned in 2.4) (+) or without AHCY inhibitor (3-deazaneplanocin A) at 1 μM. Cells were harvested by centrifuging at 2000×g for 10 min at 4 °C and the cell pellet was homogenized on ice in 1–2 mL cold PBS. This homogenized suspension was centrifuge at 10,000×g for 15 min at 4 °C, and the supernatant was removed and stored at −80 °C prior to measurement. Quantification of intracellular SAH concentration was conducted using an S-Adenosylmethionine (SAM) and S-Adenosylhomocysteine (SAH) ELISA Combo Kit (STA-671-C; from Cell Biolabs, INC. (catalogue #STA-671-C); Generon; Berkshire, UK) following the manufacturer's protocol.