Hiapp

The hIAPP stocks were prepared as previously described [39 (link)]. To this end, 1 mg of hIAPP (BACHEM AG, Bubendorf, Switzerland) was dissolved in 512 µL 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, Thermo Fisher, Kandel, Germany) and stored for 1 h at room temperature with occasional vortexing. After centrifugation at 15,000 rpm in a microcentrifuge (VWR International, Darmstadt, Germany), the upper ~80% of the solution was divided into 30-microliter aliquots. HFIP was evaporated overnight in a fume hood to obtain the dried peptide films. After HFIP evaporation, the dried aliquots were stored at −21 °C until further use. Since the current experiments required comparatively small amounts of hIAPP, the dry peptide films were redissolved in 75 µL HFIP each, vortexed for 30 s, centrifuged for 15 s using an Eppendorf MiniSpin centrifuge (Eppendorf, Hamburg, Germany), and divided into 10-microliter aliquots. These 10-microliter aliquots were dried again as described above and stored at −21 °C. Immediately before each experiment, a dried hIAPP aliquot was slowly brought to room temperature and subsequently dissolved in 10 µL dimethyl sulfoxide (DMSO, Sigma-Aldrich, Steinheim, Germany), vortexed for 30 s, centrifuged for 15 s, and allowed to reach equilibrium for 10 min.

Pancreatic β-cells (RINm5F β-cells) were obtained from the ATCC (Manassas, VA, USA). hIAPP (Sigma-Aldrich, St. Louise, MO, USA) was dissolved in dimethylsulfoxide. Fetal bovine serum (FBS) and 1% antibiotics (penicillin and streptomycin) were purchased from Gibco (Carlsbad, CA, USA). The used primary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Cell Signaling Technology (Beverly, MA, USA). 2‘,7‘-Dichlorofluorescein diacetate (DCF-DA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). All other agents were of the highest grade available unless otherwise stated.