Bsa fraction 5

INS-1 832/13 cells (kindly donated by Dr. C. B. Newgaard, Duke University, USA) were cultured in RPMI-1640 containing 11.1 mM d-glucose and supplemented with 10% fetal bovine serum, 100 U ml⁻¹ penicillin (Gibco), 100 μg ml⁻¹ streptomycin (Gibco), 10 mM N-2 hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), 2 mM glutamine, 1 mM sodium pyruvate, and 50 μM β-mercaptoethanol (Sigma), at 37 °C in a humidified atmosphere containing 95% air and 5% CO₂.
EndoC-βH1 cells (EndoCells, Paris, France) were maintained in a culture medium containing: DMEM (5.6 mM glucose), 2% BSA fraction V (Roche), 10 mM nicotinamide (Merck), 50 µM 2-mercaptoethanol, 5.5 µg ml⁻¹ transferrin, 6.7 ng ml⁻¹ sodium selenite (Sigma), 100 U ml⁻¹ penicillin, and 100 µg ml⁻¹ streptomycin (PAA Laboratories). For β-TC6 cell culture see below.

C1qA peptide (Biotin-GSKGEQGEPGAPGI, GenScript, Piscataway, NJ, USA), control peptide (Biotin-KAEQAEPAAPAI, GenScript), sDDR (2538-DR; R&D Systems), or BSA (Roche, Basel, Switzerland) were coated on an ELISA plate (Corning) at 8 μg/mL in PBS and incubated overnight at 4 °C. The plates were washed with 0.05% PBS with Tween-20 (PBST) twice and blocked with 1% BSA fraction V (Roche) in PBS at 25 °C for 1 h. Various concentrations of sDDR2 (0.1, 0.5, 2, or 8 μg/mL) or C1qA peptide (0.1, 0.5, 2, or 8 μg/mL) were added and incubated at RT for 2 h. After washing with 0.05% PBST, alkaline phosphatase (AP)-conjugated anti-6x-His Ab (3D5, Invitrogen, Waltham, MA, USA) for sDDR2 detection or AP-conjugated streptavidin (7105–04, Southern Biotech, Birmingham, AL, USA) for C1qA peptide or control peptide were incubated at RT for 1 h. Phosphatase substrate (S0942, Sigma-Aldrich) solution was added for color development, and the optical density of each well was measured at 405 nm.

EndoC-βH3 cells^{59 (link)} were maintained on a 2 µg ml⁻¹ fibronectin- and 1% extracellular matrix-coated plate in Dulbecco’s modified Eagle medium (DMEM) low glucose (1 g l⁻¹), sodium pyruvate (Thermo Fisher Scientific), 2% BSA Fraction V (Roche), 1% heat-inactivated foetal bovine serum (FBS; Labtech), 2 mM l-glutamine, 5.5 μg ml⁻¹ human transferrin, 1 mM sodium pyruvate 10 mM nicotinamide, 6.7 ng ml⁻¹ sodium selenite, 50 μM β-mercaptoethanol, 100 U ml⁻¹ penicillin and 100 µg ml⁻¹ streptomycin. DMEM was substituted with Advance DMEM/F-12 (Thermo Fisher Scientific) and FBS was omitted for the TetOn-HNF1A EndoC-βH3 cell line, as well as during the expansion of EndoC-βH3 clones.
293FT cells (Thermo Fisher Scientific) were maintained in DMEM, 10% heat-inactivated FBS, 0.1 mM MEM non-essential amino acids, 2 mM l-glutamine, 1 mM sodium pyruvate, 500 µg ml⁻¹ geneticin, 100 U ml⁻¹ penicillin and 100 µg ml⁻¹ streptomycin.
MIN6 cells^{60 (link)} were maintained in DMEM, 4.5 g l⁻¹ glucose, 15% heat-inactivated FBS, 50 μM β-mercaptoethanol and 50 µg ml⁻¹ gentamicin.

EndoC-βH1 cells (EndoC-βH1 cells, Paris, France)⁸⁸ (link) were grown in Matrigel fibronectin-coated (100 μg/mL and 2 μg/mL, respectively, Sigma–Aldrich, Steinheim, Germany) culture vessels in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) containing 5.6 mM glucose, 2% BSA fraction V (Roche Diagnostics, Mannheim, Germany), 10 mM nicotinamide (Merck Millipore, Darmstadt, Germany), 50 μM 2-mercaptoethanol, 5.5 μg/mL transferrin, 6.7 ng/mL sodium selenite (Sigma–Aldrich), 100 U/mL penicillin, and 100 μg/mL streptomycin (PAA Laboratories, Pasching, Austria). The cells were incubated in a humidified atmosphere with 5% CO² at 37°C. The cells were tested for mycoplasma contamination on a regular basis.

The human clonal β-cell line EndoC-βH1 (EndoCells, Paris, France) was seeded in 24 well plates at a density of 1.8 × 105 cells/well and maintained in DMEM culture medium (5.5 mM glucose) 2% BSA fraction V (Roche, Basel, Switzerland), 10 mM of nicotinamide (Merck, Darmstadt, Germany), 50 µM of 2-mercaptoethanol, 5.5 µg/mL of transferrin, 6.7 ng/mL of sodium selenite (Sigma), 100 U/mL of penicillin, and 100 µg/mL of streptomycin (PAA Laboratories, Toronto, Ontario, Canada) as described in [28 (link)]. For measurement of insulin secretion, the cells were cultured for 12h in the culture medium containing 2.8 mM glucose before incubation at 1 or 20 mM of glucose.