Preparing and calibrating microparticles for cell adhesion assays with the radial-flow detachment assay using the streptavidin/biotin system.

Format:

Book

Thesis/Dissertation

Author/Creator:

Majzoub, Samar S.

Contributor:

Quinn, John A., advisor.

University of Pennsylvania.

Language:

English

Subjects (All):

Chemical engineering.

Analytical chemistry.

0486.

0542.

Penn dissertations--Chemical engineering.

Chemical engineering--Penn dissertations.

Local Subjects:

Penn dissertations--Chemical engineering.

Chemical engineering--Penn dissertations.

0486.

0542.

Physical Description:

290 pages

Contained In:

Dissertation Abstracts International 60-11B.

System Details:

Mode of access: World Wide Web.

text file

Summary:

Receptor-mediated cell adhesion to ligand-coated surfaces under hydrodynamic flow is a central phenomenon in many physiological processes as well as biotechnological and medical applications. Initial studies with receptor-coated latex beads (Cozens-Roberts et al. 1990) confirmed a dynamic model for the detachment process, revealing quantitative measures of the underlying receptor/ligand interactions using complementary antibodies. Our objective is to extend these studies and develop a simple fluorescence model system to establish a "gold standard" which can be used to investigate cell adhesion behavior as means of characterizing surface shear stresses in ill-defined dynamic flow fields. We do this by preparing suitably coded model "cells", incorporating receptor-coated latex beads having known adhesion properties to ligand-coated polystyrene surfaces, and labeled with identifying fluorescent tags. These particles are calibrated with the Radial-Flow Device Assay (RFDA), which has been developed, modified and proven to be a suitable and novel technique for exploring a number of particle adhesion related phenomena. The RFDA generates a continuous range of well-characterized shear stresses within a single experiment, and is provided with an automated fluorescence microscopy and image analysis system for bead counts acquisition. The biotin/streptavidin system is widely employed in many branches of biotechnology, and was used to simulate the receptor/ligand interactions in the present work.

We perform detachment experiments with the focus on the effect of ligand and receptor densities on strength of adhesion. We analyze our experimental data and demonstrate their validation with a theoretical model initially developed by Dembo et al. (1988) that was derived from a mechanical force balance on a peeling interface. We determine the detachment forces necessary for beads displacement and compare with data reported in the literature. We further use hydrodynamic shear stress to assess the binding characteristics of various bead populations, expressing different receptor numbers, to variable ligand densities. We suggest and demonstrate how these results can be used to separate beads on the basis of their differential adhesiveness to specified ligand-coated substrate surfaces, and how this separation is influenced by shear force and ligand density. We further show how such results might be useful for probing surface shear fields in an unknown environment.

Notes:

Thesis (Ph.D. in Chemical Engineering) -- University of Pennsylvania, 1999.

Source: Dissertation Abstracts International, Volume: 60-11, Section: B, page: 5647.

Adviser: John A. Quinn.

Local Notes:

School code: 0175.

ISBN:

9780599558694

Access Restriction:

Restricted for use by site license.