Annv pe

To further explore betamethasone effect, NIT-1 and isolated islets cells were cultured with CM with or without betamethasone (100 nM, Sigma-Aldrich) for 2–10 days. CM was replaced every 2-3 days.
At least 5 × 10⁵ NIT-1 cells were cultured for 2 days with graded concentrations of betamethasone (up to 10 μM) or CM. Cells were harvested using 0.05% trypsin EDTA (ThermoFisher) and stained for anti-CD44 BV786 (BD Biosciences), anti-Class I MHC eFluor 450 (eBioscience), AnnV PE (Immunotools) and 7aad (BD Biosciences). Median Fluorescence Intensity (MFI) and viability were assessed using flow cytometry (LSR Fortessa, BD Biosciences). Corresponding fluorescence minus one staining was used as control. Data were analysed using FlowJo software (Tree Star Inc.). Culture supernatant from NIT-1 cells was frozen at −80 °C until use. Insulin secretion was assessed by measuring C-peptide concentration in the supernatant by ELISA (RayBiotech, Norcross, GA, USA).

To study the effect of betamethasone, spleens from prediabetic NOD mice (8–10 weeks of age) were harvested and single-cell suspensions were obtained by mechanical disruption. After haemolysis, cell number and viability were determined by AnnV PE (Immunotools), 7aad (BD Biosciences) and Perfect Count Microspheres (Cytognos SL., Salamanca, Spain) using flow cytometry (FACS Canto II, BD Biosciences). Splenocytes were cultured at 10⁶ cells/mL with 0.1 nM, 1 nM, 2.5 nM, 5 nM, 10 nM, 50 nM, 100 nM or 1000 nM betamethasone or the corresponding controls (PBS). Cell viability was determined as abovementioned at 24 h using flow cytometry. Human PBMCs were obtained from 10 mL blood sample by means of Ficoll Paque (GE Healthcare, Marlborough, MA, USA) density gradient centrifugation. PBMCs were cultered at 10⁶ cells/mL with 0.1 nM to 10⁶ nM betamethasone. After 48 h, cell viability was determined in PBMCs using flow cytometry as detailed above, and three leukocyte subsets were analyzed using anti-CD3 APC, anti-CD14 APC-Cy7 and anti-CD19 PE (Immunotools).

NIT-1 cells were stained with anti-CD44 BV786 (BD Biosciences), anti-class I major histocompatibility complex (MHC) eFluor-450 (eBioscience), anti-CD14 PE, and anti-CD49b FITC (Immunotools). Viability was assessed with AnnV PE (Immunotools) and 7aad (BD Biosciences) as detailed above. Median fluorescence intensity and viability were determined using flow cytometry (LSR Fortessa, BD Biosciences). Corresponding fluorescence minus one staining was used as control. The data were analyzed using FlowJo (Tree Star Inc).