A flow cytometric method to measure the stimulated mobilization and the intracellular pool of the adhesion promoting glucoprotein Mac-1.

The receptor for complement factor C3bi (Mac-1 or CR3) belongs to a complex of leukocyte surface glucoproteins (CD11/CD18) that are essential for chemotaxis and adhesion of human polymorphonuclear leukocytes (PMN). Granulocytes can increase their surface expression of Mac-1 upon stimulation and it is proposed that this depends on a rapid mobilization of an intracellular pool of Mac-1. In the present study we describe a cell membrane permeabilization method that enables the detection of the intracellular pool of Mac-1 in granulocytes by flow cytometry. The method is based on the use of the non-ionic detergent n-octyl-beta-D-glucopyranoside (OG) to permeabilize the cell membranes of paraformaldehyde-prefixed leukocytes. It is shown that fMLP (5 x 10(-7) M)-treated cells expose 85% of the total detectable amount of Mac-1 molecules on the surfaces. The method makes it possible to measure the total detectable pool, the efficiency of Mac-1 mobilization and the in vivo expression of the receptor. This could be of value when evaluating the role of adhesion proteins in the inflammatory response.