Time-resolved fluorometric assay for leukocyte adhesion using a fluorescence enhancing ligand

Area of Science:

• Biomedical Assays
• Cellular Immunology
• Fluorescence Spectroscopy

Background:

• Leukocyte adhesion is a critical process in inflammation and immune response.
• Accurate and efficient measurement of leukocyte adhesion is essential for research and diagnostics.
• Existing methods, such as microscopic counting, can be time-consuming and labor-intensive.

Purpose of the Study:

• To develop and validate a novel time-resolved fluorometric assay for quantifying leukocyte adhesion in vitro.
• To optimize the labeling and assay conditions for isolated human neutrophils.
• To compare the performance of the new assay with traditional microscopic counting methods.

Main Methods:

• Development of a 96-well microtiter plate assay utilizing a fluorescence-enhancing ligand, 2,2′:6′,2″-terpyridine-6,6″-dicarboxylic acid (TDA).
• Leukocytes were loaded with TDA, which accumulates intracellularly after esterase hydrolysis.
• Adherent cells were lysed, releasing TDA to form a fluorescent chelate with europium, measured by time-resolved fluorometry.

Main Results:

• The assay demonstrated a direct correlation between fluorescence signal and the number of adherent leukocytes.
• Optimized TDA-labeling and adhesion assay conditions were established for human neutrophils.
• The fluorometric assay showed comparable or superior performance to microscopic counting in terms of speed and reproducibility.

Conclusions:

• The developed time-resolved fluorometric assay provides a rapid, reproducible, and convenient method for routine analysis of leukocyte adhesion.
• This assay offers a significant advancement over traditional methods for studying leukocyte adhesion in vitro.
• The technique has potential applications in immunological research, drug screening, and diagnostic development.