Mechanical Measurement And Instrumentation Control By A K Sawhney Pdf !!LINK!! Download

Cell-matrix mechanical interactions play a defining role in a range of biological processes such as developmental morphogenesis and wound healing. Despite current agreement that fibroblasts exert mechanical forces on the extracellular matrix (ECM) to promote structural organization of the collagen architecture, the underlying mechanisms of force generation and transduction to the ECM are not completely understood. Investigation of these processes has been limited in part by the technical challenges associated with simultaneous imaging of cell activity and measurement of cellular forces.

Although all of these experimental models have provided important insights into cell mechanical behavior, they are limited by the fact that silicone is a non-physiological substrate. This limitation has recently been overcome, in part, by the use of flexible polyacrylamide sheets embedded with fluorescent microspheres as a substratum (Pelham and Wang, 1997; Pelham and Wang,1999; Wang and Pelham,1998). Unlike silicone, the surface of these sheets can be coated with ECM proteins, and the material property of the sheet can be controlled by varying the acrylamide/bis-acrylamide ratio. Polyacrylamide sheets have been used to study the magnitude and pattern of force generation during locomotion of normal and H-ras transformed 3T3 fibroblasts(Munevar et al., 2001a), to determine the response of cells to gradients in substrate rigidity(Pelham and Wang, 1997), and to study the effects of myosin inhibitors on the pattern of force generation(Pelham and Wang, 1999).

Mechanical Measurement And Instrumentation Control By A K Sawhney Pdf Download

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For expression of GFP-zyxin, human zyxin in a pEGFP-N1 vector (Clontech laboratories, Palo Alto, CA) was used. This probe was a generous gift of Professor J. Wehland and coworkers (BGF, Braunschweig, Germany), and has been used previously as a marker for focal contacts in goldfish fin fibroblasts(Beningo et al., 2001; Kaverina et al., 2000; Kaverina et al., 1999) and mouse melanoma cells (Rottner et al.,2001). The unmodified pEGFP-N1 expression vector was used as a control. Only first or second passage corneal fibroblasts were used for transfections, since we have found previously that cell mechanical activity is reduced after multiple passages. Corneal fibroblasts were plated on six-well plates (40,000 cells/well) using complete media without antibiotics 24 hours before transfection. Transfection was performed using Lipofectamine PLUS(Invitrogen, Carlsbad, CA). For each well, 0.5 g of GFP-zyxin DNA in 100l DMEM was pre-complexed with 3 l PLUS reagent for 15 minutes. Meanwhile, 2.5 l Lipofectamine reagent was mixed with 100 l DMEM. Pre-complexed DNA and diluted Lipofectamine reagent then were mixed and incubated for 15 minutes at room temperature. Cells were then incubated with DNA-PLUS-Lipofectamine reagent complex mixed with an additional 800 l of DMEM (total of 1 ml of media per well). After 3 hours of incubation at 37C (5% CO2), 1 ml of DMEM and 20% FBS was added to each well. After 24 hours, transfection media was replaced with complete media.

Eighteen hours after plating on the gel, most cells (both GFP-zyxin transfected and controls) had a bipolar morphology similar to in vivo wound healing corneal fibroblasts. The cell density was sparse enough to focus on the mechanical activity of isolated cells. DIC imaging allowed detailed visualization of the fibrillar collagen organization in addition to the beads and cells (Fig. 2A). GFP-zyxin was organized into adhesions that were most concentrated at the leading edge and tail of the cell (Fig. 2B),although adhesions were also present along the ventral surface of the cell body. In general, these adhesions were slightly below those at the ends of the cell, and thus were not always visualized in the same focal plane. Adhesions at the leading edge were generally organized into a radial pattern. Diffuse background GFP-zyxin labeling was also present, suggesting a cytoplasmic pool of this protein. Control cells transfected with the EGFP-N1 vector alone had bright and diffuse non-specific cytoplasmic and nuclear labeling, with no specific labeling of adhesions (not shown). e24fc04721