Quantitation of polymorphonuclear leukocyte migration under agarose by enzyme assay

Migration of polymorphonuclear leukocytes under agarose is widely used to assay the mobility of these cells. We modified this assay so that directed and random migration of polymorphonuclear leukocytes might be quantitated rapidly and objectively by an enzyme method. The optimal method used gelatin-agarose (0.25% gelatin; 0.875% agarose) gel layered on glass coverslips. At the end of the migration period, the leukocytes which had not migrated under the agarose were removed by aspiration and washing. The remaining leukocytes were solubilized with Triton X-100 and the myeloperoxidase activity on the coverslips determined. With appropriate standards, this activity could be used to estimate the number of polymorphonuclear leukocytes which had migrated in a random or in a directed (chemotactic) manner. Excellent correlation was observed between the myeloperoxidase method and direct microscopic counting of the number of cells that had migrated. The sensitivity of the assay could be enhanced by inducing bidirectional migration under 2 simultaneous gradients of chemotactic factor situated 180 degrees apart. This assay is objective, reproducible and combines the simplicity of assessing migration by measuring distance of the 'leading cell front' with the accuracy of individual cell counting.