μ slide 6 0.4 channels

A bespoke setup was established consisting of 5% CO₂/air supplied from a gas cylinder (BOC) to OB1 Microfluidic flow controller (Elveflow) to control the air pressure applied to the input of a μ‐Slide I luer 0.4 or μ‐Slide VI 0.4 channels (ibidi) containing cells and media, while the output is closed with a stopper. Particular attention was paid to leave no airspace between the stopper and the media. Cells were seeded at 22,000 cells per channel and maintained in 1% FBS complete DMEM throughout the duration of the experiments. Cyclic hydrostatic pressure at 0.1 Hz of 100 mbar or 200 mbar was applied to cells.

Cells were seeded as described for hydrostatic pump experiments in μ‐Slide VI 0.4 channels (ibidi) and maintained at 37°C, 5% CO₂, overnight. Cells were serum‐starved for 1 h then incubated on ice with pre‐cooled 100 µg/ml dextran Oregon green 488 (Life Technologies). Dextran uptake was stimulated using pre‐warmed dextran Oregon green 488 and 100 ng/ml human recombinant EGF (Gibco) in serum‐free DMEM for 10 or 30 min at 37°C in conjunction with hydrostatic pressure or at cells at steady state.

Cells were seeded as described for hydrostatic pump experiments in μ‐Slide VI 0.4 channels (ibidi) and maintained at 37°C, 5% CO₂, overnight. Cells were serum starved for 1 h then incubated on ice for 10 min in pre‐cooled 50 µg/ml transferrin‐Alexa 594 (Life Technologies). Transferrin uptake was stimulated by incubating cells at steady state with pre‐warmed 50 µg/ml transferrin‐Alexa 594 at 37°C or in conjunction with hydrostatic pressure. Surface labelling of transferrin was removed by washing cells with ice‐cold stripping buffer (29.2 g/l NaCl, 0.5% (v/v) acetic acid in distilled H₂O) twice for 30–40 s.