Application of hydrostatic pressure to cells in vitro was accomplished as previously described (5 (link)–7 (link)) using modifications. Briefly, cells were seeded into Opticell (Thermo Scientific, Rochester, NY) cassettes and allowed to adhere overnight. Pressure was created by connecting the Opticell to an IV bag filled with cell culture media utilizing arterial line tubing. Opticell’s were clamped between perforated stainless steel sheets secured by binder clips. Hydrostatic pressure was modulated by adjusting the height of the IV bag. Pressure was determined using the following formula: P=ρ·h; where P=hydrostatic pressure, ρ=fluid density, and h=the height of the IV bag in relation to the Opticell.
Viability was assessed by pressurizing cells for 24 hours and counting live cells by trypan blue staining. CM was isolated from MLO-Y4 cells by pressurizing cells for 24 hours in RPMI containing 0.1% FBS at 0, 20, and 40 mmHg. CM was prepared as described above. BCA assay was used to quantify protein concentration of cells; no significant (p>0.05) differences were observed after normalization.
Viability was assessed by pressurizing cells for 24 hours and counting live cells by trypan blue staining. CM was isolated from MLO-Y4 cells by pressurizing cells for 24 hours in RPMI containing 0.1% FBS at 0, 20, and 40 mmHg. CM was prepared as described above. BCA assay was used to quantify protein concentration of cells; no significant (p>0.05) differences were observed after normalization.
