Kinetic titration method in flow cytometry for quantification of cell receptors

Area of Science:

• Immunology
• Biophysics
• Analytical Chemistry

Background:

• Accurate quantification of cell surface receptors is crucial for understanding cellular function and disease.
• Traditional methods for receptor quantification using fluorescence flow cytometry have limitations, including reliance on calibration beads and potential inaccuracies due to reaction kinetics.

Purpose of the Study:

• To develop and validate a novel, calibration-bead-free method for quantitative determination of cell receptors using fluorescence flow cytometry.
• To account for reaction kinetics in receptor-ligand binding for more accurate cell surface receptor quantification.

Main Methods:

• A new method based on reaction kinetics was proposed, involving samples with constant cell concentration and varying initial fluorescently labeled ligand concentrations.
• Each sample was measured by flow cytometry when the product of incubation time and initial ligand concentration was constant across all samples.
• The method was applied to quantify CD8 and CD14 receptors on human peripheral blood leukocytes.

Main Results:

• The proposed method successfully quantified CD8 and CD14 receptors without using calibration beads.
• A significant decrease (two-fold) in the mean number of CD14 receptors was observed on monocytes and granulocytes of atherosclerosis patients compared to healthy donors.
• No significant difference in mean CD8 expression was found on leukocytes between the patient and donor groups.

Conclusions:

• The developed kinetic method offers an alternative approach for accurate cell receptor quantification by flow cytometry, overcoming limitations of existing methods.
• The findings suggest a potential role for reduced CD14 expression in the pathophysiology of atherosclerosis.
• The method provides a valuable tool for immunological research and clinical diagnostics.