Au x0550

Embryonic rat cardiac H9c2 (2-1) cells (ECACC, Salisbury, UK) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Aurogene, Rome, Italy), containing 5.5 mM d-glucose and supplemented with 10% heat-inactivated fetal bovine serum (FBS; AU-S181H Aurogene, Rome, Italy), 1% L-Glutamine (L-Glu; AU-X0550 Aurogene, Rome, Italy), and 1% penicillin/streptomycin solution (P/S; AU-L0022 Aurogene, Rome, Italy), at 37 °C under an atmosphere of 5% CO₂.
After reaching 80% confluence, H9c2 cells were trypsinized, seeded at a specific cell density for each assay, and then exposed to NG, high glucose (HG; 33 mM d-glucose), or NG + 27.5 mM mannitol (M; as an osmotic control) for 48 h [40 (link)]. Cells were then treated for 6 days [41 (link)] in NG or HG medium with the following substances:-

CHR 0.399 mg/mL (CHR), dissolved in NaCl;

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SBECD 7.3 m/m%, dissolved in NaCl;

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Binary system SBECD + 0.095 mg/mL CHR (SBECD + CHR), dissolved in NaCl;

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DMSO 2.5% as a vehicle of OTX008;

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OTX008 (0.75–1.25–2.50 µM);

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Binary system OTX008 (2.5–1.25–0.75 µM)-SBECD (OTX008-SBECD), dissolved in NaCl;

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Ternary system CHR (0.324 mg/mL)-OTX008 (2.5–1.25–0.75 µM)-SBECD (CHR-OTX008-SBECD), dissolved in NaCl.

Three independent experiments were conducted, each performed in triplicate (N = 9).

Human colonic epithelial cells (HCoEpC; iXCells Biotechnologies) were cultured in Epithelial Cell Growth Medium (iXCells Biotechnologies, Cat# MD-0041) and all experiments were performed by seeding 5 × 10⁴ HCoEpC /well in 6-well plates. B95–8, an EBV positive marmoset cell line, was cultured in RPMI 1640 (Sigma-Aldrich, St Louis, MO, USA, R0883), 10% fetal bovine serum (FBS; Sigma-Aldrich, F7524), 2 mM glutamine (Aurogene, Rome, Italy, AU-X0550), 100 mg/ml streptomycin and 100 U/ml penicillin (Aurogene, AU-L0022), in 5% CO₂-saturated humidity at 37 °C. All experiments were performed with mycoplasma-free cells.
Human peripheral blood mononuclear cells (PBMCs) were obtained from buffy coats of healthy donors through lymphocyte cell separation medium (Cedarlane, CL5020) and B lymphocytes were isolated by immunomagnetic cell separation using anti-CD19-conjugated microbeads, according to the manufacturer's instructions (Miltenyi Biotec, 130–050–301). B cells were cultured in complete medium, in 5% CO₂-saturated humidity at 37 °C.

As previously described (Hermenean et al., 2023 (link)), embryonic rat cardiac H9c2 (2-1) cells (ECACC, United Kingdom) were cultured at 37°C under an atmosphere of 5% CO₂, in Dulbecco’s modified Eagle’s medium (DMEM; Aurogene, Italy). This growth medium contained 5.5 mM D-glucose, 1% L-Glutamine (L-Glu; AU-X0550 Aurogene, Italy), 10% heat inactivated fetal bovine serum (FBS; AU-S181H Aurogene, Italy) and 1% penicillin/streptomycin (P/S) solution (AU-L0022 Aurogene, Italy). H9c2 cells were seeded at a specific density for each assay before being exposed to NG, high glucose (HG; 33 mM D-glucose) or NG + 27.5 mM mannitol (M; as osmotic control) for 48 h (Hermenean et al., 2023 (link)). Cells were then treated in NG or HG medium for 6 days (Hermenean et al., 2023 (link)) with the following substances:

- CHR 0.399 mg/mL dissolved in NaCl (CHR);

- SBECD 7.3 m/m% dissolved in NaCl (SBECD);

- SBECD + 0.095 mg/mL CHR dissolved in NaCl (SBECD + CHR);

- as vehicle for OTX008, dimethyl sulfoxide 2.5% (DMSO);

- OTX008 (0.75–1.25–2.50 µM);

- SBECD-OTX008 (2.5–1.25–0.75 µM) dissolved in NaCl (SBECD + OTX);

- SBECD-OTX008 (2.5–1.25–0.75 µM)-CHR dissolved in NaCl (SBECD + OTX + CHR).

Three independent experiments were done, each performed in triplicates (N = 3). Cell morphology was observed at the optical microscope.