Cellasic onix2 microfluidic system

Exponentially growing cells at OD_{600 nm} = 0.2 were injected into the microfluidics chambers of a CellASIC plate (Y04C-02 plate for haploid cells, CellASIC ONIX2 Microfluidic System; Millipore), through the inlet wells. A constant flow of rich media (20 µl/h for 0.036-µl chambers) fed the chambers, which were kept at the constant temperature of 32°C. Cells in the microfluidic device were imaged using a fully motorized Axio Observer Z1 inverted microscope (Zeiss) with a 100× immersion objective, a Hamamatsu Orca R2 camera, and constant focus maintained with focus stabilization hardware (Definite focus, Zeiss). The temperature was maintained at 32°C with a controlled heating unit and an incubation chamber. Images were acquired every 10 min using ZEN software (Zeiss). All aspects of image acquisition were fully automated and controlled, including temperature, focus, stage position, and time-lapse imaging.

For osmotic stabilization experiments, we used both custom-made and commercially-available setups. Briefly, we used the commercially-available CellASIC ONIX2 Microfluidic System (Merck, Germany) as follows. Bacteria were grown by diluting an overnight culture 1:200 in fresh LB to an OD₆₀₀ of between 0.1 and 0.2 and incubating at 37°C. The bacterial solution was loaded into the appropriate channels using the manufacturer's pre-set loading sequence. After loading, the solution was immediately exchanged to LB+cephalexin (50 μg/mL) to induce cell lysis. At the onset of bulging, the LB+cephalexin solution was switched to LB+cephalexin+sorbitol to stabilize bulges, and the channels were continuously supplied with fresh LB+cephalexin+sorbitol at a flow rate of ~0.2 μL/h (corresponding to a set pressure of 0.5 kPa in the CellASIC system).
We also used simple, custom-made microfluidic setups comprising rectangular channels with lowered centers. These devices were designed in AutoCAD (Autodesk, San Rafael, CA), fabricated using in-house UV lithography, and replicated in polydimethylsiloxane (PDMS) by soft lithography (Weibel et al., 2007 (link)), as described previously (Renner and Weibel, 2011 (link)).