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.
Lundahl
J
J
Dept. Clinical Immunology, Karolinska Hospital, Stockholm, Sweden.
Halldén
G
G
Hed
J
J
Johansson
S G
SG
D000293:Adolescent; D000328:Adult; D000368:Aged; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D005960:Glucosides / Q000494:pharmacology; D006098:Granulocytes / Q000276:immunology; D006801:Humans; D016177:Macrophage-1 Antigen / Q000032:analysis* / Q000276:immunology; D008875:Middle Aged; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D010539:Permeability
D005960:Glucosides; D016177:Macrophage-1 Antigen; C018619:octyl-beta-D-glucoside; D009240:N-Formylmethionine Leucyl-Phenylalanine