Heat inactivated fcs

RINm5F Insulinoma cells (ATCC, Wesel, Germany) were maintained in RPMI 1640 supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin (Life Technologies, Darmstadt, Germany), and 10% heat-inactivated FCS (Biochrom, Berlin, Germany) at 37°C and 5% CO₂. For experiments, cells were seeded on 6-well and 96-well polystyrene plates (Greiner, Frickenhausen, Germany) in aforementioned culture medium.
Murine islets of Langerhans were isolated from wildtype C57Bl/6J mice and cultured at 37°C and 5% CO₂ in RPMI 1640 supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 1% non-essential amino acids, 1% pyruvate (Life Technologies), and 10% heat-inactivated FCS (Biochrom). Briefly, the pancreas was dissected and perfused with collagenase P solution (1.2 U/ml in RPMI 1640, Roche Applied Science, Mannheim, Germany). Islets were isolated by density gradient centrifugation using Ficoll Paque Plus (GE Healthcare, München, Germany), were washed, and handpicked thereafter. Isolation by combined density gradient centrifugation and subsequent handpicking as performed herein results in highly pure cultures with islet purity >99% (Ramírez-Domínguez and Castaño, 2015 (link)). After 48–96 h recovery in aforementioned medium, 60–100 islets (as specified in the respective figure legends) were seeded on 12-well polystyrene plates (Greiner, Frickenhausen).

The murine oropharyngeal cell lines (MOPCs) were kindly provided by J. Lee (Sanford Research/University of South Dakota) and cultured in medium containing DMEM high glucose and Ham's F12 (3:1) (Thermo Fisher Scientific, Karlsruhe, Germany) supplemented with 10 % (v/v) heat-inactivated FCS (Biochrom, Berlin, Germany), 100 IU ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin (Thermo Fisher Scientific), epidermal growth factor (5 μg ml⁻¹) (Biochrom), insulin (5 μg ml⁻¹), transferrin (5 μg ml⁻¹), cholera toxin (0.0084 μg ml⁻¹), hydrocortisone (0.5 μg ml⁻¹) and tri-iodo-thyronine (0.00136 μg ml⁻¹) (Sigma-Aldrich, Taufkirchen, Germany)51 (link). MOPC^−eGFP cells were generated by lentiviral gene transfer using a pCL6IEGwo empty vector52 (link) and enriched to >90 % enhanced green fluorescent protein (eGFP) positivity by FACS analysis. The murine lung carcinoma cell line TC1 was kindly provided by Z. Fridlender (Hadassah Medical Center, Israel) and the murine bladder cancer cell line MB49 by K. Esuvaranathan (University of Singapore, Singapore). Both cell lines were cultured in DMEM high glucose supplemented with 10 % (v/v) FCS, 100 IU ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin and 1 mM sodium pyruvate (Thermo Fisher Scientific). All cells were routinely tested for mycoplasma contamination using the Venor Mycoplasma Detection Kit (Minerva Biolabs, Berlin, Germany).