Lumox cell culture dishes

For electron microscopy (EM), cells were cultured on lumox cell culture dishes (Sarstedt AG & Co., Nümbrecht, Germany) and fixed in 4% PFA, 2.5% glutaraldehyde, and 0.1 M cacodylate buffer (pH 7.4) for 15 min at 37 °C. After PBS wash, cells were fixed with 1% OsO4 in cacodylate buffer for 1 h at RT followed by a second wash with 0.1 M cacodylate buffer. Cells were dehydrated using an acetone series and mounted in Epon resin (Fluka, Buchs, Switzerland). Blocks with embedded cells were sectioned with a vibratome VT 1000 S (Leica, Wetzlar, Germany) and ultrathin sections were stained with uranyl acetate and lead citrate. The sections were examined and photographed with a Zeiss EM10 electron microscope (Zeiss) and a Gatan SC1000 OriusTM CCD camera (GATAN, Munich, Germany) in combination with the DigitalMicrograph™ 3.1 software (GATAN, Pleasanton, CA, USA).

To experimentally separate mechanotransductive and hypoxic effects on macrophages that occur concomitantly during OTM, we used lumox cell culture dishes (94.6077.331, Sarstedt), with oxygen-permeable membranes, so that the adherently growing cells could still be supplied with oxygen under experimental pressure application, as pressure and hypoxic conditions are induced concomitantly by the glass disc applied (Figure 1). Approximately 250,000 RAW264.7 macrophages per ml were seeded either onto conventional polystyrene plates (353046, Omnilab) or on lumox plates. After 24 h of preincubation, macrophages were either compressed using glass plates with a defined weight of 2 g/cm² according to an established and published model [7 (link), 16 (link), 17 (link)] or left untreated for further 24 h (Figure 1). To correspond to the results from repeated Experiment 1 with the addition of DMOG, we also repeated this experiment with compressive force treatment for 4 h (Supplemental figures 8-11).