Pen strep solution

HeLa cells were purchased from ATCC (Cat # CCL‐2) and cultured at 37°C, 5% CO₂ in DMEM, high glucose (Life Technologies, Cat # 41966), supplemented with 10% FBS (Life Technologies, Cat # 10270106), and 1% Pen/Strep solution (Sigma‐Aldrich, Cat # P4333). Same medium and conditions were employed for HCT‐116 cells (Clontech, Cat #630931) whereas HuH‐7 cells, received from the Health Science Research Resources bank of the Japan Health Sciences Foundation (Cat #JCRB0403), were grown in GlutaMAX (Life Technologies, Cat # 21885‐025), supplemented with 10% FBS (Life Technologies, Cat # 10270106) and 1% Pen/Strep solution (Sigma‐Aldrich, Cat #P4333).

Cultures of midbrain dopaminergic neurons on top of cortical astrocytes were made from P1-P2 Wistar rats or DAT-IRES-Cre mice (Jackson Laboratory)^{36 (link)}. Briefly, tissue was dissected from the ventral midbrain and digested in a papain solution oxygenated with a carbogen (95% O₂ + 5% CO₂) at 37 °C for 30 min. The digested tissue was brought to a single cell suspension by trituration through pipette tips of increasingly smaller sizes and centrifuged at 500 g for 10 min. The neurons were resuspended in a prewarmed neuron medium (Neurobasal A (10888022, Gibco) with 1% GlutaMAX (35050061, Gibco), 2% B-27 plus (A3582801, Gibco), 200 µM ascorbic acid, 500 µM kynurenic acid, and 0.1% Pen-Strep solution (P0781, Sigma)).
The cells were plated in neuron medium in six-well plates on a monolayer of glia cells grown on poly-d-lysine coated coverslips (⌀ = 25 mm). Two hours after plating neurons, rat glial cell-derived neurotrophic factor (SRP3239, Sigma) was added for a final concentration of 10 ng/mL. The cultures were used for experiments 14–21 days after the neurons were plated out. The neurons obtained from DAT-IRES-Cre mice were transduced 5 days post dissection with pAAV-FLEX-tdTomato (Addgene).

Preadipocytes were isolated from iWAT and eWAT stroma vascular fraction (SVF) and differentiated in an adipogenic culture media after two passages to eliminate non‐preadipocyte cell contamination. In detail, WAT was aseptically minced and incubated with collagenase Type II (1 mg/mL, Sigma) at 37℃ for 45 min. After digestion, serum‐containing medium was added to the suspension and filtered through a 100 μm cell strainer. SVF cells were centrifuged for 10 min at 350 g and suspended in pre‐warmed adipogenic culture media, consisting of DMEM high glucose with Glutamax (Gibco) supplemented with 10% newborn calf serum (Thermo fisher), 2.4 nM human insulin (Sigma), and 1% antibiotic solution (Pen‐Strep solution, Sigma). To induce differentiation, SVF cells were seeded at near‐confluence and medium was replenished every day to allow selection by adhesion during the first 3 days and then changed every 2 days. SVF cells were cultured in an atmosphere of 5% CO₂ and 20% O₂ at 37℃. Based on preliminary experiments, day 7 was the time of differentiation used for all experiments.