Immobilization of nonactivated unfixed platelets for real-time single-cell analysis

Abstract

Existing methods for measuring the response of individual platelets to stimulation are limited. They either measure each platelet at one discrete time-point (flow cytometry) or rely on adhesive ligands to immobilize platelets that concomitantly generate activation signals (microscopy). Such methods of immobilization make it impossible to assess resting platelets, the changes that occur as platelets transition from resting to active states, or the signals generated by soluble agonists, such as ADP and thrombin, or by mechanical stimulus, independently from those generated by the adhesive ligand. Here we describe a microscopy method that allows the immobilization of platelets to a glass cover slip without triggering platelet activation. This method makes use of specific antibodies that bind platelet PECAM-1 without activating it. Platelets can therefore be immobilized to PECAM-1 antibody coated biochips without causing activation and perfused with agonists or inhibitors. Using this method, platelets can be stimulated by an array of soluble agonists at any concentration or combination, in the presence or absence of inhibitors or shear forces. This chapter describes in detail this PECAM-1 mediated immobilized platelet method and its use for measuring changes in Ca 2+ signaling in individual platelets under a number of different conditions. While we focus on the measurement of Ca 2+ dynamics in this chapter, it is important to consider that the basic method we describe will easily lend its self to other measures of platelet activation (integrin activation, shape change, actin dynamics, degranulation), and may, therefore, be used to measure almost any facet of platelet activation.