Plan fluor 40 objective

The PCB hosting the device was placed in a custom-made holder, which fits onto the stage of an automated, inverted microscope (Figure S 3b). The images were obtained using inverted microscopes (Olympus IX 81 and Nikon Ti Eclipse microscope) placed in an environmental control box, which maintained a stable temperature of 30 °C. Fluorescence images were captured on the Nikon microscope using a Nikon Plan Fluor 40× objective (NA 0.75, WD 0.66). The microscope was controlled using Youscope^{28 (link)}, and offline image analysis was performed using ImageJ^{33 (link)} and CellX³⁴ . Syringe pumps (neMESYS, Cetoni GmbH, Germany) were connected to the microfluidic chip for media supply (flow rate 10 µl/min, 15 ml of medium is required for a 24-h experiment) and were controlled using Youscope.
Experiments to assess the medium exchange characteristics (Figure S 4) were carried out on the Olympus microscope, and images were taken every second with a CMOS camera (Hamamatsu ORCA Flash 4.0 camera). Syringes were filled with de-ionized water and a solution of Amaranth (4 mg/ml, Sigma-Aldrich, Switzerland) in de-ionized water. Switching between the different syringes was controlled using Youscope. The calculation of the average intensity of the images and further analysis was performed using ImageJ.

Immunostaining was performed as described previously [12 (link)]. In brief, after treatment, cells were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized for 15 min with 0.2% Triton X-100 (Merck Millipore, 94101-L), and then blocked with 5% goat serum for 1 h. they were subsequently incubated with primary antibodies overnight at 4 °C followed by incubation for 1 h with secondary antibodies at room temperature. Their nuclei were stained with a DAPI solution. Images were captured with a fluorescent microscope (Nikon Eclipse 80i equipped with Nikon PLAN FLUOR × 40 objective) or Nikon confocal microscope (Nikon, N-STORM and A1). Photographic images were resized and analyzed by ImageJ software. The following primary antibodies were used for immunostaining: SQSTM1 (Santa Cruz Biotechnology, sc-28359); K48-linked Ub chain-specific antibody (Merck Millipore, 05-1307); FLAG Tag (Prospec, ANT-146); Phospho-SQSTM1 (S403)-specific antibody (Cell Signaling Technology, 39786), Alexa Flour 488- or 568-conjugated secondary antibodies (Thermo Fisher Scientific, A11034, A11029, A11031, A11036); See Additional file 1: Table S2 for further details and dilutions of all antibodies.