Testing the influence of hyperglycemia on the content of amino acids, fatty acids and selected lipids in extracellular vesicles using tof-sim

A human pancreatic β-cell line (1.1B4) was maintained in RPMI 1640 medium supplemented with 10% foetal bovine serum (FBS), 1% 200 mM L-glutamine and antibiotics - 100 μg/mL streptomycin and 100 units/mL penicillin. Half of the dishes were cultured under NG conditions (11 mM D-glucose), while the second half was grown under long-term (3 passages) HG conditions (35 mM D-glucose). Collected conditioned media were centrifuged to remove cell fragments and debris . Then, supernatants in a volume of 300 mL were concentrated using the 1000 kDa (MWCO - molecular weight cut-off) dialysis cellulose membranes (Spectra/Por Biotech) at low vacuum (−0.3 Bar) to obtain 1 ml of the EV sample. In the next step, the concentrated sample was centrifuged to remove apoptotic bodies. The supernatant was divided into two Eppendorf tubes, which were then subjected to various fractions of EVs: mixed fraction of EVs, ectosomes fraction and exosomes fraction.