Flexercell strain unit fx 3000

Briefly, cyclic TF was applied on MC3T3-E1 cells with the Flexercell Strain Unit (FX-3000, Flexcell Corp.) [Shimizu et al., 1995 (link)]. The Flexercell Strain Unit was used in order to mechanically strain cells. MC3T3-E1 cells were seeded on flexible-bottomed six-well plates that have a hydrophilic surface at a density of 2 × 10⁴ cells/cm², then cells were placed onto a vacuum manifold controlled by computer software and a solenoid valve. The system uses a vacuum source to apply a negative pressure causing a downward deformation of the membrane to which the cells are attached. The strain applied over the loading-post regions is approximately equal in the radial and circumferential directions [Vande Geest et al., 2004 (link)]. Cells were flexed at 6 cycles/min (5 s strain, 5 s relaxation) with 6% or 18% TF for maximum 24 h. We selected the same TF strength as referenced elsewhere [Shimizu et al., 1995 (link)]. Controls were prepared in an identical manner and cultured on unstrained flexible-bottomed plates.

Primary cultures of neonatal rat ventricular myocytes were prepared from 2- to 4-day-old Sprague-Dawley rats, as described previously^{20 ,27 ,55 (link)}. For stretch experiments, primary cardiomyocytes were cultured on collagen I-coated elastomere plates with flexible bottom (Bioflex, Flexcell) in density of 0.15 million cells per cm². Stretch was introduced to attached cardiomyocytes by a computer-controlled vacuum suction with Flexercell Strain Unit FX-3000 (Flexcell), as described previously^{30 (link)}. Frequency of cyclic stretch was 0.5 Hz with pulsation of 10–25% elongation of cells for 24 h. Small molecules 3i-1000 and 3i-0777 in supplemented CSFM media containing 0.1% DMSO were added into the cells 1 h before starting the mechanical stretching. For the control samples, the cells were plated and cultured similarly on collagen I flexible bottom culture plates without stretching.
For phenylephrine experiments, primary cardiomyocytes were cultured on normal cell well plates (BD Falcon) at the density of 0.13–0.17 million cells per cm². On the third day on culture, the cells were first exposed to 3i-1000 in supplemented CSFM media containing 0.1% DMSO and then 1 h later, 100 μM phenylephrine (Sigma) was added into the media for 24 h.

The cells were exposed to cyclic mechanical stretch for 1, 4, 12, 24 or 48 hours by applying a computer controlled (Flexercell Strain Unit FX-3000, Flexcell International Corporation) vacuum suction under the flexible-bottomed collagen I-coated 6-well cell culture plates, as previously described [22] . After experiments the cells were quickly frozen with nitrogen oxide at −70°C.