New chamber for flow cytometric analysis over an extended range of stream velocity and application to cell adhesion measurements.

Abstract

When analyzed in a flow cytometer, particles are suddenly accelerated to high velocities (1-10 m.s-1) over very short distances. This feature is essential to obtain high analysis rates and low coincidence levels, but translates into very strong velocity gradients (greater than 10(5) s-1): particles experience strong hydrodynamic stresses that elongate them and tend to dissociate weakly associated complexes. In order to analyze fragile conjugates formed by heterotypic adhesion between two cell types, a flow cytometer was modified to make hydrodynamic stress not only much weaker but also adjustable. A new and easily adaptable flow cell was designed for the instruments of the FACS series; it provided satisfactory hydrodynamic conditions on a wide continuous range of flow rates. Accompanying electronic adaptations permitted standard analysis between 0.01 and 10 m.s-1. At 0.01 m.s-1, the velocity gradient roughly amounts to 50 s-1. Conjugates formed by the adhesion between human B and resting T lymphocytes, disrupted in conventional flow cytometers, could be detected and precisely quantified provided analysis velocity was kept below 0.1 m.s-1. We conclude that low velocity flow cytometry makes possible the quantification of weak intercellular adhesion phenomena, and is potentially useful for the future development of new biomechanical techniques and other applications.