Bovine serum albumin (bsa)

Resuspended in 100 µL PBS + 1% BSA (Equitech-Bio, Kerrville, TX, USA) + 1:1000 kathon CG/ICP (Supelco, Bellefonte, PA, USA) staining buffer with primary and secondary antibodies was 1 × 10⁷ CFU of (r)LA, as listed in Supplementary Table S5. Samples were analyzed on a Gallios flow cytometer (Beckman Coulter, Indianapolis, IN, USA). Analyses were performed in FlowJo (v.10.8.1, Ashland, OR, USA) with gating based on NCK56 and secondary antibody-only controls.

Cryopreserved islet preparations were thawed by rapid (150°C/min) warming to 4°C followed by the removal of DMSO with a sucrose gradient and serial dilution of cryoprotectant [11 ]. The islets were subsequently cultured overnight in CMRL 1066 (Corning, Tewksbury, MA, USA) supplemented with 0.5% BSA (Equitech-Bio, Kerrville, TX, USA), 1% Insulin-Transferrin-Selenium (Corning), 100 U/mL penicillin/streptomycin (Life Technologies, Burlington, ON, Canada) and l-glutamine (Sigma-Aldrich, Oakville, ON, Canada). Both the cryopreserved and the freshly isolated islets were cultured for an additional 24 h in low-glucose (1 g/L) DMEM supplemented with 10% FBS and 100 U/mL penicillin/streptomycin.

Cells were rinsed twice with 37°C PBS and fixed with 1% formaldehyde (Sigma) in PBS for 10 min. Cells were permeabilized with PBS containing 0.1% Triton X-100 (Sigma) for 10 min and blocked with TBS containing 5% BSA (Equitech-Bio) and 0.1% Triton X-100. A mouse antibody against vimentin (clone V9, Santa Cruz Biotech sc-57678) was diluted in TBS containing 5% BSA and 0.1% Triton X-100 and incubated with the samples overnight at 4˚C. Cells were washed three times with PBS and then incubated with a goat anti-rabbit (H + L) Alexa Fluor 647-conjugated secondary antibody (Thermo Fisher) diluted in TBST containing 5% BSA for 1 hr at room temperature. Coverslips were washed five times with PBS and mounted on slides using ProLong Diamond anti-fade mounting medium with DAPI (Thermo Fisher). Cells were imaged using a confocal laser scanning microscope (LSM 880; Zeiss) equipped with an automatic stage, Airyscan detector (Hamamatsu) and diode (405 nm), argon ion (488 nm), double solid-state (561 nm), and helium-neon (633 nm) lasers. Images were acquired using a 60x/1.4 NA oil objective (Zeiss) and deconvolved using automatic Airyscan Processing in the Zen Software (Zeiss).

Embryonic explants were dissected from 10.5-dpc mouse embryos and were incubated in DMEM/F12 (without glucose, pyruvate and phenol red (Cell culture technologies), supplemented with 0.5mM glucose and 1% BSA (Equitech-Bio) at 37°C, 5% CO₂, 60% O₂ for 30 min. Explants were then dissected into posterior and anterior PSM fragments in equilibrated medium and were then transferred to a calibrated Clarke type oxygen electrode (Hansatech Instruments) maintained at 37°C. Basal oxygen consumption rate was measured for posterior and anterior PSM fragments (n=20 per reading) and then normalized to the total cell number.

Culture experiments were done as described previously(Lauschke et al., 2013 (link)). Mouse embryos were collected at 10.5-dpc in PBS with 1% BSA (Equitech-Bio) and 0.5mM glucose. PSM explants consisted of the entire PSM plus three pre-formed somites. Samples for the 2D PSM ex vivo cultures were prepared as described (Lauschke et al., 2013 (link)). The culture medium was DMEM/F12 (without glucose, pyruvate and phenol red, Cell culture technologies), supplemented with 0.5mM Glucose and 1% BSA, if not specified otherwise. Additional metabolites were added in the concentrations indicated in the experiments. Explants were cultured in pre-equilibrated culture medium in 8-well chamber slides (Lab-Tek #155411) in a humidified chamber at 37°C, 5% CO₂ and 60% O₂ (PSM explant cultures) and 37°C, 5% CO₂ in ambient air (2D PSM ex vivo cultures).

When fully anesthetized, a tracheostomy was performed to maintain ventilation and anesthesia during thoracotomy. The aorta was exposed and cannulated. Hearts were quickly dissected, transferred to the Langendorff setup (Hugo Sachs) and perfused at ~80 mmHg with a 37°C warm Krebs–Henseleit (KH) buffer (in mmol/L: 118 NaCl, 4.7 KCl, 1.2 KH₂PO₄, 1.2 MgSO₄7H₂O, 1.75 CaCl₂2H₂0, 25 NaHCO₃ and 11 glucose), equilibrated with carbogen (95% O₂ ‐ 5% CO₂). To assess contractile function, a latex balloon connected to a pressure transducer (Hugo Sachs Electronic) was placed in the left ventricle via the left atrium. A 37°C buffer filled bath was placed around the hearts. The hearts were perfused for 10 min in KH buffer followed by 110 min perfusion with either a palmitate‐glucose KH buffer: KH buffer + 1.2 mmol/L palmitic acid, 1.5 mmol/L Na₂CO₃, 3% bovine serum albumin (BSA) (Equitech‐Bio, Inc. Kerrville, Texas), or a glucose KH buffer: KH buffer + 3% BSA. BSA:palmitate buffer was prepared as described in (Lopaschuk and Barr 1997). Coronary flow and left ventricular developed pressure (LVDP) were recorded using IOX 2 (EMKA technologies), from which heart rate (HR), maximum increase in pressure over time during contraction (dP/dt_max), and maximum decrease in pressure over time during relaxation (−dP/dt_max) were derived.