Purification of pericytes from rodent optic nerve by immunopanning

Abstract

This protocol describes the use of immunopanning to purify rodent pericytes from the optic nerve. Immunopanning permits the prospective isolation of pericytes from optic nerve tissue by relying on the binding of pericytes to an anti-PDGFRβ (platelet-derived growth factor receptor beta) antibody adhered to a Petri dish. The cells are viable at the end of this gentle procedure, and they can be analyzed acutely for gene expression or cultured alone or in coculture with other central nervous system (CNS) cell types, including CNS endothelial cells and CNS astrocytes. As written, this procedure is used for isolation of optic nerve pericytes from the rat. The same PDGFRβ antibodies can be used for purifying optic nerve pericytes from the mouse, but alternate negative panning antibodies must be used to ensure that astrocytes do not contaminate the preparation. This procedure can also be modified to purify pericytes from the brain. The same PDGFRβ antibody is used, but additional steps (specific dissections or negative panning) are required to ensure that other PDGFRβ-positive contaminants, including cells from the rostral migratory stream, are depleted from the cell suspension.