Selenite, Sodium

Cryopreserved PHH (obtained from KalyCell, France or Bioreclamation IVT, USA) or fresh hepatocytes (obtained from patients subject to liver resections at Huddinge University Hospital, Stockholm, Sweden) were used for formation of spheroid cultures. Hepatocytes obtained from patient livers were isolated as previously described42 (link). Use of the liver specimens for this purpose was approved by the Ethics Committee at Karolinska Institutet (Regionala etikprövningsnämnden i Stockholm) and studies were carried out in accordance with the approved guidelines. Written informed consent was obtained from all donors of liver material. Cells were seeded into ultra-low attachment 96-well plates (Corning) at 1,500 viable cells per well and subsequently centrifuged at 100 × g for 2 min. Cells were seeded in 100 μl Williams E medium supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 10 μg/ml insulin, 5.5 μg/ml transferrin, 6.7 ng/ml sodium selenite, 100 nM dexamethasone, and 10% FBS. Spontaneous self-aggregation of the hepatocytes initiated spheroid formation. From day 4 or 5 after seeding, when the spheroids were sufficiently compact, 50% of the medium was exchanged daily for serum-free medium. Spheroids were maintained in serum-free medium until day 35, with a medium change every 48–72 h.

The immortalized erythroid progenitor cell lines were induced to differentiate into more mature erythroid cells by culture in erythroid differentiation medium; IMDM (Sigma) containing 10% human AB serum (Kohjin Bio, Saitama, Japan or TAKARA BIO), α-tocopherol (20 ng/ml; Sigma), linoleic acid (4 ng/ml; Sigma), cholesterol (200 ng/ml; Sigma), sodium selenite (2 ng/ml; Sigma), holo-transferrin (200 µg/ml; Sigma), human insulin (10 µg/ml; Sigma), ethanolamine (10 µM; Sigma), 2-ME (0.1 mM; Sigma), D-mannitol (14.57 mg/ml; Sigma), mifepristone (an antagonist of glucocorticoid receptor, 1 µM; Sigma) and EPO (5 IU/ml).

DITNC1 astrocytes were seeded at a density of 5 × 10⁵ cells/cm² and incubated to 90% confluency in 6-well glass bottom plates. Then, the cells were treated with OMVs (2.5 μg/ml) or TNF (10 ng/ml) for 12 h or 48 h, respectively. Next, the medium was eliminated, and cells were washed twice with PBS and fixed with 4% paraformaldehyde for 15 min. The reaction was then stopped with 0.1 mM glycine. Following extensive washing with PBS, CAD cells labeled with Cell Tracker Green CMFDA (10 µM) were seeded over fixed astrocytes at a density of 1 × 10⁴ cell/cm² in DMEM F12 media, supplemented with 8% FBS and antibiotics [55 (link)]. Before 24 h of culture, the media was replaced with fresh SFM containing 50 ng/ml sodium selenite (S5261, Sigma-Aldrich) to induce morphologically visible neuronal differentiation. After 24 h, neuronal processes were captured with an Epifluorescent Spinning disk microscope (Olympus). The neurite extension length was determined using the NeuronJ plug-in of the v1.8 NIH ImageJ Software, as previously described [56 (link), 57 (link)].

Five thousand cells/well were plated in 96-well Ultra Low Attachment coated round bottom plates (Corning, UK). After spheroid formation (usually after 24 h), 100 μL of fresh medium containing a 2X concentration of sodium selenite (Na₂SeO₃) or SeNPs (BSA or chitosan coated) were added. Cell viability was determined using a CellTiterGlo assay (Promega, UK). For the viability assay, an increasing dose range (0.01 μg/mL to 20 μg/mL selenite of SeNPs) was applied by dilution in the appropriate medium for 24, 48 or 72 h. After the treatment, 100 μL of media was removed from wells and 100 μL of CellTiterGlo added. Plates were shaken for 5 min and equilibrated at room temperature for 25 min before luminescence measurements were taken (BMG Labtech Fluostar Omega, UK). IC20 and IC50 doses were determined as the concentration required to reduce the luminescence signal by 20/50%. The IC20/50 values shown are the result of a minimum of five independent experiments performed with 4 technical repeats. IC50 values calculated as 1.25 μg/mL for SeNP-BSA and 3 μg/mL for SeNP-chitosan, which were only slightly less than that for selenite (IC50 0.6 μg/mL) in SKOV-3 spheroids. Whereas in OVCAR-3 models the IC50 was calculated as 5 μg/mL for both SeNP-BSA and SeNP-chitosan compared to selenite (IC50 10 μg/mL). IC20 values calculated as 0.15 μg/mL for SeNP-BSA and 0.15 μg/mL for SeNP-chitosan, which were slightly lower than that for selenite (IC20 0.3 μg/mL) in SKOV-3 spheroids. Whereas in OVCAR-3 models the IC20 was calculated as 0.6 μg/mL for both SeNP-BSA and SeNP-chitosan compared to selenite (IC20 0.3 μg/mL).

Bovine ovaries from randomly cycling cattle and other endocrine tissues (pituitary glands, testes and adrenal glands) were obtained from an abattoir. Ovaries were dissected to obtain antral follicles ranging in diameter from 3 mm to 18 mm, and these were further processed to isolate the GC layer, TC layer, and follicular fluid, as described previously (Glister et al. 2010 (link)). Briefly, follicles were sorted into five different size classes: 3–4 mm (n = 8), 5–6 mm (n = 8), 7–8 mm (n = 9), 9–10 mm (n = 6) and 11–18 mm (n = 12). Each follicle was hemisected, and GC and TC layers were recovered for RNA extraction, while follicular fluid was recovered for steroid hormone analysis. Large follicles (11–18 mm) were reclassified according to their oestrogen to progesterone ratio (E:P ratio) in follicular fluid as either large oestrogen-active (LEA; E2:P4 ratio >1) or large oestrogen-inactive (E2:P4 ratio <1) follicles. Corpora lutea (CL) at growing (n = 4), mid-luteal (n = 5) and regressing (n = 4) stages were also harvested. All tissue samples were homogenized in Trizol reagent for total RNA extraction, as described previously (Glister et al. 2010 (link)).
Follicles (4–8 mm diameter) were also retrieved for isolation of GC and TC to be used for primary cell culture experiments, as described in detail elsewhere (Glister et al. 2001 (link), 2005 (link)). GC and TC were seeded into 96-well plates (Nunclon, Life technologies Ltd.) at a density of 75,000 cells/250μL/well for serum-free culture (non-luteinized cells) or 10,000 cells/250 μL/well for serum-supplemented culture (luteinized cells). Cells were cultured for 6 days at 38.5°C with saturating humidity in 5% CO₂ in air. The culture medium consisted of McCoy’s 5A medium (Sigma), supplemented with antibiotic/antimycotic solution (1% v/v; Sigma), apo-transferrin (5 μg/mL; Sigma), sodium selenite (5 ng/mL; Sigma), bovine insulin (10 ng/mL; Sigma), HEPES (20 mM; Sigma) and bovine serum albumin (0.1% w/v; Sigma). Medium used for serum-free GC culture was also supplemented with 10^–7 M androstenedione as aromatase substrate. For GC and TC cultured under conditions that promote luteinization, 2% fetal calf serum (FCS) was also included as a supplement. In all four culture models, media were changed after 48 h and replaced with fresh media containing treatments as specified below. This was repeated after a further 48-h incubation period. Media were retained after the final 48-h period (i.e. 96–144 h) for subsequent analysis of steroid hormone secretion. Viable cell number at the end of culture was determined by neutral red uptake, as described elsewhere (Glister et al. 2001 (link)).
It should be noted that culturing TC and GC using defined serum-free medium preserves a non-luteinized phenotype reflected by LH-induced androstenedione (A4) secretion by TC and follicle-stimulating hormone (FSH)-induced oestradiol (E2) secretion by GC. Henceforth, these cells will be referred to as non-luteinized TC (NLTC) and non-luteinized GC (NLGC). In contrast, culturing TCs and GCs under serum-supplemented conditions promotes spontaneous luteinization, as indicated by reduced A4/E2 secretion and greatly increased secretion of P4 (Glister et al. 2001 (link), 2005 (link), Kayani et al. 2009 (link)). Henceforth, these cells will be referred to as LTC and LGC.