Ham s f12

PC3 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in DMEM/Ham’s F12 medium, i.e. a mixture (1:1) of Dulbecco’s Modified Eagle’s Medium: Ham’s F12 (Gibco^®, Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin (Lonza, Basel, CH). Cells were maintained at 37 °C in humidified atmosphere supplied with 5% CO₂. Cell harvesting was performed by a Trypsin/EDTA treatment as per the manufacturer’s protocol (Sigma-Aldrich).

The human GPR44-expressing Chinese hamster ovarian (CHO-K1) stable cell line was purchased from PerkinElmer and was cultured, according to the provider’s recommendations, in Ham’s F-12 (Gibco) supplemented with 10% FBS (Gibco), 0.4 mg/mL G418 (Corning), 1% Penicillin/Streptomycin (PEST) (Gibco) and 2 nM L-Glutamine (Gibco). Non-transfected CHO-K1 cells used as negative control were purchased from ATCC and cultured in Ham’s F-12 (Gibco) supplemented with 10% FBS (Gibco) and 1% PEST (Gibco).
The human beta-cell line EndoC-BH1 was obtained from Endocells and cultured according to the provider’s indications in DMEM low glucose supplemented with 2% Bovine Serum Albumin (BSA) (Roche Diagnostics), 50 µM 2-mercaptoethanol (Sigma-Aldrich), 10 mM nicotinamide (VWR Life Science), 5.5 µg/mL transferrin and 6.7 ng/mL sodium selenite.

Primary myoblasts were isolated according to previously published methods [60 (link)]. Briefly, excess fat and blood was quickly removed from fresh muscle samples (~50 mg), and samples were minced in trypsin-EDTA (0.25%; Gibco, ThermoFisher Scientific, Waltham, MA, USA) 1:4 in Ham’s F-12 (Cytiva, Fisher Scientific, Pittsburgh, PA, USA) and agitated twice for 45 min each in 10 mL total (5 mL per agitation) of the trypsin-EDTA/Ham’s F12 mixture at room temperature. After adding 2 mL of fetal bovine serum (FBS, MilliporeSigma, Burlington, MA, USA), the mixture was centrifuged (500× g, 5 min, room temperature), supernatant was separated, and the cell tissue mixture was resuspended in growth medium containing 10% FBS in Ham’s F-12 with 2.5 ng/mL fibroblast growth factor (FGF, 233-FB, R&D systems, Bio-Techne, Minneapolis, MN, USA), 1× penicillin streptomycin (1% of final volume; Gibco, ThermoFisher Scientific, Waltham, MA, USA), and 4 mM L-glutamine (Gibco, ThermoFisher Scientific, Waltham, MA, USA). The cells were incubated overnight (37 °C, 5% CO₂) before moving to a new culture dish to remove fibroblasts. The growth medium was changed every other day. The cells were grown to approximately 40–50% confluence for passage (P)0 with dense (~100% confluent) individual colonies and 80–90% confluence for subsequent passages. Experiments were performed with myoblasts at P4.

Small intestine was harvested from 5-week-old mice, cut into small pieces, embedded in a matrix, and cultured. The method was performed as previously published [15 (link)], with exception of the composition of the acellular bottom layer and cell-containing layer, which were prepared according to the protocol for organotypic cell culture [14 (link)]. The culture was performed in a transwell (24 mm transwell with 3.0 μm pore polycarbonate membrane insert, Corning) that was placed into a 6-well transwell carrier (Organogenesis). The culture medium was composed of Ham's F12 (Life Technologies), 20% FBS (Life Technologies), and 50 μg/mL Gentamicin (Life Technologies) and it was exchanged after one week. 1 mL of medium was added per well. After two weeks, the culture was fixed in formalin and embedded in paraffin and PAS staining was performed.