Vybrant dio lipophilic dye

NMVMs were plated on micropatterned substrates for stretch, and incubated with 5 μM Vybrant DiO lipophilic dye (Molecular Probes, Life Technologies, Grand Island, NY) for 3 days from plating. Fluorescence was excited using a Lambda DG-4 fluorescent light source and captured with Photometrics Cascade 512F camera using MetaMorph v6.1 acquisition software. Fluorescent intensity per unit cell area was measured in the same cells before and immediately after 14% longitudinal by 3.6% transverse stretch. Each observation consisted of 6–20 cell measurements.

N-type [100]-orientation silicon wafers with 1933 nm silicon oxide (Addison Engineering) were cut into ~0.5 cm² chips using a diamond pen. Chips were cleaned with warm 20% 7× detergent, then washed with copious amounts of 18 MΩ water. After cleaning, chips were placed into wells of an 8-well Nunc Lab-Tek II chambered coverglass. Bilayers were prepared on the chips using the same procedures as for coverslip supported bilayers. To measure bilayer heights, liposomes for supported lipid bilayers were labelled with 200:1 Vybrant DiO lipophilic dye (Molecular Probes) in PBS for 5 minutes, and then applied to the chips. Imaging was performed on an inverted Ti-E Perfect Focus System (Nikon) controlled by Metamorph software, equipped with 488 nm and 561 nm lasers, a motorized laser Ti-TIRF-E unit, a 1.49 NA 100× TIRF objective, emCCDcamera (QuantEM 512; Photometrics), and with a linear glass polarizing filter (Edmunds Optics) in the excitation laser path. Preparation of SAIM calibration wafers, and SAIM imaging and analysis were performed as described previously (35 (link)). All images were filtered with a 1 pixel σ Gaussian filter to smooth background noise. For quantitative image analysis, local background subtraction followed by intensity thresholding was used to create whole cell and microcluster masks.