Electrochemiluminescence technology

The mycoplasma-free alpha TC1–6 cell line was purchased from ATCC (CRL-2934; Barcelona), and maintained in DMEM (ThermoFisher Scientific) supplemented with 10% FBS, 15 mM HEPES, 0.1 mM non-essential amino acids, 0.02% BSA, 2 g/l glucose, 1.5 g/l sodium bicarbonate and 5 µM beta mercaptoethanol (all purchased from Sigma-Aldrich). The proliferation of splenocytes isolated from mouse spleens was assessed after a 3-day culture in RPMI 1640 medium supplemented with 8% FBS, 20 mM l-glutamine, 1% sodium pyruvate, 1% nonessential amino acids, and 1% penicillin/streptomycin (all from Invitrogen), in the presence or absence of the insulin peptide SLYQLENYCA. Cells were pulsed with [³H]-thymidine for the last 24 h of culture, harvested and lysed onto membranes prior to liquid scintillation counting using a Beckman Coulter LS 6500 counter. Mouse primary macrophages were isolated from the peritoneal cavity, and cultured in DMEM/F12–10 (ThermoFisher Scientific) supplemented with 10% FBS, 2 mM l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin (all purchased from Sigma-Aldrich). Cells were stimulated with 1 μg/ml LPS (in DMSO), in the absence or presence of 0.1, 1, or 10 μM BL001 for 24 h. The secretion of cytokines was measured in the culture medium by electrochemiluminescence technology from MesoScale Discovery (Rockville, USA), and RNA was extracted from cells.