Endogenous component chemotactic assay (ECCA).

Abstract

We have developed a chemotactic assay in which migrated cells are quantitated by measuring levels of an endogenous cellular component. The endogenous component chemotactic assay (ECCA) employs standard double-membrane, blind-well methodologies but is unique in that leukocyte migration is quantitated by measuring lactic dehydrogenase (LDH) activity endogenous to cells that have migrated. This approach avoids the tedium of microscopic counting as well as the problems associated with cell-labeling techniques. Using the ECCA technique we have shown: (1) that N-formylmethionyl-leucyl-phenylalanine (fMLP) is both chemokinetic and chemotactic for human polymorphonuclear neutrophils (PMNs); (2) that both incubation time and starting PMN density affect the proportion of cells that migrate; (3) that approximately 30% of the available PMNs eventually migrate; and (4) that PMN "fall off" from membranes, readily detectable by this assay, is affected by starting PMN density, incubation period, and nature of the attractant. The technique as presented can detect migration when a starting cell density as low as 7 x 10(4) PMNs/well is employed and can be made more sensitive by increasing the period over which LDH is allowed to act. Considerable potential exists to further apply the ECCA concept to the study of the migration of subpopulations of cells in mixtures by assaying for distinguishing endogenous cellular markers.