Anti cd44 bv786

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).

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).