Magnetic Twisting Cytometry.

Dynamic changes in cell stiffness were measured in isolated human ASM using forced motions of functionalized beads anchored to the cytoskeleton through cell surface integrin receptors, as previously described in detail (Fabry et al., 2001; An et al., 2006; Deshpande et al., 2010). The increase or decrease in stiffness is considered an index of single-cell smooth muscle contraction and relaxation, respectively. For these studies, serum-deprived, postconfluent cultured ASM cells were plated at 30,000 cells/cm² on plastic wells (96-well Removawell, Immulon II; Dynatec Labs, El Paso, TX) previously coated with type I collagen (VitroCol; Advanced BioMatrix, Inc., San Diego, CA) at 500 ng/cm², and maintained in serum-free media for 24 hours at 37°C in humidified air containing 5% CO₂. These conditions have been optimized for seeding cultured cells on collagen matrix and for assessing their mechanical properties (Fabry et al., 2001; An et al., 2006; Deshpande et al., 2010). For each individual cell, the baseline stiffness was measured for the first 60 seconds, and after drug addition, the stiffness was measured continuously for the next 240 seconds. Drug-induced changes in cell stiffness approached a steady-state level by 240 seconds. Unless otherwise stated, ASM cells were pretreated for 5 minutes with 3 μM pepducin. The effects of the inhibitors were normalized to respective steady-state drug effects (without inhibitors).