μ slide 1 0.6 luer

Human Umbilical Vein Endothelial Cells (HUVECs) were isolated and cultured as described previously [16 (link)]. The culture medium was composed of medium 199 (Gibco, Bleiswijk, the Netherlands), supplemented with 20% (v/v) fetal bovine serum, 50 μg/ml heparin (Sigma-Aldrich), 12.5 μg/ml endothelial cell growth supplement (Sigma-Aldrich) and 100U/ml penicillin/streptomycin (Gibco). All culture surfaces were fibronectin coated.
Cells were subjected to laminar shear stress as previously described [4 (link)], with the following modifications: cells were seeded in a parallel plate type flow chamber [μ-slide I 0.6 luer (Ibidi, Martinsried, Germany) coated with fibronectin and exposed to a calibrated mean shear stress level of 18 dyne/cm² during 5 days.

For experiments involving application of an acute bolus, recordings were carried out under perfused media conditions, as for Crosby et al. (2017) . Cells were seeded and grown to confluence in a microfluidics slide (μ-Slide I 0.6 Luer, Ibidi) in high glucose (27.8 mM), glutamax-containing DMEM (GIBCO) with 10% serum (HyClone FetalClone III) and pen/strep. Cultures were incubated for 1-2 weeks, with DMEM media refreshed every 5-7 days. Cells were also subjected to temperature entrainment cycles during this time. Before the start of recording, cells were changed to low-glucose (5.5 mM) DMEM (Sigma) supplemented with 1% glutamax, pen/strep and 1 mM luciferin. Cells were then perfused with the same media, driven by syringe pump (NE-1600) at a flow rate of 50 μl/hr for the duration of the experiment. Recording was performed using an ALLIGATOR (Cairn Research). Boli were applied using a diversion of flow, with no change in flow rate or detectable flow disturbance.