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

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Abstract
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 times 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.

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