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Endogenous component chemotactic assay (ECCA).

H R Creamer, W L Gabler, W W Bullock

    Inflammation
    |December 1, 1983
    PubMed
    Summary
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    A new assay quantifies cell migration by measuring lactic dehydrogenase (LDH) in migrated leukocytes. This method accurately measures polymorphonuclear neutrophil (PMN) chemotaxis and chemokinesis without cell labeling.

    Area of Science:

    • Immunology
    • Cell Biology
    • Biochemistry

    Background:

    • Leukocyte migration is crucial in immune responses.
    • Traditional methods for quantifying cell migration, such as microscopic counting or cell labeling, are often tedious and problematic.
    • There is a need for a more efficient and reliable method to study chemotaxis.

    Purpose of the Study:

    • To develop and validate a novel chemotactic assay for quantifying leukocyte migration.
    • To assess the utility of the Endogenous Component Chemotactic Assay (ECCA) for studying polymorphonuclear neutrophil (PMN) behavior.
    • To investigate the effects of incubation time, cell density, and attractants on PMN migration.

    Main Methods:

    • Developed the Endogenous Component Chemotactic Assay (ECCA) using standard blind-well methodologies.

    Related Experiment Videos

  • Quantified leukocyte migration by measuring lactic dehydrogenase (LDH) activity in migrated cells.
  • Utilized N-formylmethionyl-leucyl-phenylalanine (fMLP) as a chemoattractant for human PMNs.

    • Main Results:

    • The ECCA accurately quantifies PMN migration without cell labeling.
    • N-formylmethionyl-leucyl-phenylalanine (fMLP) demonstrated both chemokinetic and chemotactic effects on human PMNs.
    • Incubation time and starting PMN density significantly influenced the proportion of migrating cells, with approximately 30% of available PMNs migrating.
    • The assay detected PMN 'fall off' from membranes, influenced by cell density, incubation period, and attractant type.
    • The ECCA can detect migration at cell densities as low as 7 x 10^4 PMNs/well and can be further enhanced for sensitivity.

    Conclusions:

    • The Endogenous Component Chemotactic Assay (ECCA) provides a sensitive, efficient, and reliable method for quantifying leukocyte migration.
    • ECCA overcomes limitations of traditional cell counting and labeling techniques.
    • The assay has significant potential for studying cell migration, including subpopulations in complex mixtures by analyzing endogenous cellular markers.