Jin-Yu Shao 1 Jinbin Xu Department of Biomedical Engineering, Washington University, Saint Louis, MO 63130 A Modified Micropipette Aspiration Technique and Its Application to Tether Formation From Human Neutrophils Tether formation, which is mechanically characterized by its threshold force and effective viscosity, is involved in neutrophil emigration from blood circulation. Using the micropi- pette aspiration technique, which was improved by quantitative contact control and com- puterized data analysis, we extracted tethers from human neutrophils treated with IL-8, PMA, or cytochalasin D. We found that both IL-8 and PMA elevated the threshold force to about twice as large as the value for passive neutrophils. All these treatments decreased the effective viscosity dramatically (80%). With a novel method, the residual cortical tension of the cytochalasin-D-treated non-spherical neutrophils was measured to be 8.8 pN/m. DOI: 10.1115/1.1486469 Introduction Tethers are long membrane tubes extracted from cell or lipo- some surfaces when a point force is imposed. Very likely, tether formation accompanies and facilitates human neutrophil rolling on the endothelium, which is a critical initial step for neutrophil migration from blood vessels to sites of infection or injury 1,2. Under physiological conditions, tethers have been observed dur- ing neutrophil rolling on a substrate coated with activated platelets or P-selectin 2. After rolling, human neutrophils adhere firmly to the endothelium before they squeeze through the junction of en- dothelial cells. Starting from the firm adhesion, neutrophils remain in their activated state until they reach their destination. Although tether formation from passive neutrophils has been studied thor- oughly, whether tethers can be formed from activated or stimu- lated neutrophils and the properties of such tethers are still un- known. Therefore, we tried extracting tethers from human neutrophils activated respectively with interleukin-8 IL-8 and phorbol 12-myristate 3-acetate PMA. To assess the role of the membrane-cytoskeleton association in tether formation, we also examined tethers extracted from neutrophils treated with cytocha- lasin D. All these compounds are used frequently in neutrophil functioning assays in vitro 3. The mechanics of tether formation is best characterized by two parameters: the threshold force, which needs to be overcome for the membrane to flow into a tether ( F 0 ), and the effective viscos- ity (  eff ). In general, the pulling force imposed on the cell or liposome surface F and the tether growth velocity ( U t ) are gov- erned by 4–6 F =F 0 +2   eff U t . (1a) F 0 is intrinsically determined by the membrane tension, the mem- brane bending modulus, and the adhesion energy between the membrane and cytoskeleton.  eff represents the energy dissipation caused by the membrane flow, the membrane interbilayer slip, and the membrane slip over the cytoskeleton 4,6. These relationships can be expressed as 6 F 0 =2  R t  T +  + B / R t (1b) and  eff =2  m + si h 2 ln R 0 / R t  + sc R t 2 ln R 0 / R t  , (1c) where R t is the tether radius, T is the far-field tension,  is the adhesion energy between the membrane and cytoskeleton, B is the bending modulus of the membrane,  m is the membrane viscosity,  si is the interbilayer slip viscosity, h is the membrane thickness, R 0 is the characteristic far-field radius, and  sc is the cytoskeletal slip viscosity. For passive human neutrophils, F 0 =45 pN and  eff =1.8 pN•s/  m, measured with the micropipette aspiration technique MAT1,7. Although the MAT has been successfully applied to the studies of neutrophil microvillus extension, neutro- phil tether formation, neutrophil single receptor anchoring strength, and endothelial cell receptor kinetics 1,8,9, its applica- tion to other studies has been limited by its coarse contact control and its time-consuming data analysis, commonly done with the manual operation of video calipers. Here, we present an improved version of the MAT that overcomes these limitations. In the MAT, a precise suction pressure (  p ) is applied across a spherical object the force transducer that fits snugly but slips freely within a micropipette. The magnitude of the force F im- posed on the spherical object can be calculated with 7 F = R p 2  p  1 - 4 3  ¯  1 - U t U f  , (2) where R p is the radius of the micropipette, U t is the velocity of the force transducer during adhesion it is equal to the tether growth velocity since one end of the tether is linked to the force transducer while the other end stays stationary, and U f is the velocity of the transducer when it is moving freely under the same pressure  p , and  ¯ =( R p -R s )/ R p where R s is the radius of the force transducer. If a spherical latex bead is used as the force transducer, it is crucial to determine the bead diameter precisely to minimize the uncertainty in the force calculation, as can be seen in Eq. 2. The accuracy of measuring small bead diameters in a liquid environment has been limited by optical diffraction. Al- though small bead diameters can be measured much more pre- cisely with electron microscopy, it is not practical to take the bead that is used in one experiment to an electron microscope and measure its diameter. Therefore, we developed a method that al- lowed us to determine small bead diameters in solution with an accuracy of about one pixel in a digitized bead image. To quanti- tatively control the contact between the force transducer and sub- strate, we built a device that enabled us to apply alternating con- stant pressures to the force transducer. We also developed two computerized tracking programs that allowed us to track the bead motion with an accuracy of about 30 nm and 5 nm respectively. 1 Address all correspondence to: Jin-Yu Shao, Ph.D., Department of Biomedical Engineering, Washington University, Campus Box 1097, 517 Lopata Hall, One Brookings Drive, St. Louis, MO 63130-4899, Email: shao@biomed.wustl.edu Contributed by the Bioengineering Division for publication in the JOURNAL OF BIOMECHANICAL ENGINEERING. Manuscript received September 2001; revised manuscript received April 2002. Associate Editor: C. Dong. 388 Õ Vol. 124, AUGUST 2002 Copyright © 2002 by ASME Transactions of the ASME Downloaded 23 Sep 2009 to 128.252.212.99. Redistribution subject to ASME license or copyright; see http://www.asme.org/terms/Terms_Use.cfm