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Updated: Aug 8, 2026
Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface
Published on: November 2, 2011
Flow cytometric analysis and modeling of cell-cell adhesive interactions: the neutrophil as a model
S I Simon1, J D Chambers, L A Sklar
1Department of Pathology, University of New Mexico, Albuquerque 87131.
Insights
Neutrophil aggregation, crucial for immune function, occurs in three phases. This study models these phases, revealing that new aggregates form mainly early on, while existing ones are stabilized by adhesive molecules.
Area of Science:
- Immunology
- Cell Biology
- Biophysics
Background:
- Intercellular adhesion is vital for immune cell function, mediated by molecules like Leu-CAMs.
- Leukocyte cell adhesion, particularly neutrophil aggregation, is a transient and reversible process.
Purpose of the Study:
- To quantitatively elucidate homotypic aggregation events in neutrophils.
- To mathematically model the kinetics of neutrophil aggregation stimulated by formyl peptides.
Main Methods:
- Neutrophil aggregation was induced by formyl peptide stimulation.
- Aggregate formation kinetics were mathematically modeled using a linear model.
- Flow cytometry and intravital stain LDS-751 were used to distinguish and quantify aggregates.
Main Results:
- Neutrophil aggregation followed a three-phase time course with distinct rate constants.
- The initial phase (30-60s) showed rapid aggregation; a plateau phase (up to 2.5 min) had reduced aggregation rates.
- A disaggregation-predominant phase (2-3 min) showed increased disaggregation rates; CD18 antibody suggested stabilization of existing aggregates.
Conclusions:
- Neutrophil aggregation kinetics can be described by a three-phase model.
- Significant new aggregate formation primarily occurs in the initial phase.
- Adhesive contact sites within aggregates may stabilize them during the plateau phase, involving molecules like CD18.
Abstract:
The immune function of granulocytes, monocytes, lymphocytes, and other specialized cells depends upon intercellular adhesion. In many cases the molecules mediating leukocyte cell adhesion belong to the Leu-CAM superfamily of adhesive molecules. To elucidate the events of homotypic aggregation in a quantitative fashion, we have examined the aggregation of neutrophils stimulated with formyl peptides, where aggregate formation is a transient reversible cell function. We have mathematically modeled the kinetics of aggregation using a linear model based on particle geometry and rates of aggregate formation and breakup. The time course was modeled as a three-phase process, each phase with distinct rate constants. Aggregate formation was measured on the flow cytometer; singlets and larger particles were distinguished using the intravital stain LDS-751. Aggregation proceeded rapidly after stimulation with formyl peptide (CHO-nle-leu-phe-nle-tyr-lys). The first phase lasted 30-60 s; this was modeled with the largest aggregation rate and smallest rate of disaggregation. Aggregate formation plateaued during the second phase which lasted up to 2.5 min. This phase was modeled with an aggregation rate nearly an order of magnitude less than that of the initial fast phase, whereas the disaggregation rate for this phase did not change significantly. A third phase where disaggregation predominated, lasted the remaining 2-3 min and was modeled with a four to fivefold increase of the disaggregation rate. The mechanism of cell-cell adhesion in the plateau phase was probed with the monoclonal antibody IB4 to the CD18 subunit of the adhesive receptor CR3. Based on these studies it appears that new aggregates do not form to a large degree after the first phase of aggregate formation is complete. However, new adhesive contact sites may form within the contact region of these adherent cells to keep the aggregates together.
