Multiparametric Characterization of Telomere Length in T Cells by Flow-FISH

Area of Science:

• Immunology
• Cell Biology
• Aging Research

Background:

• Telomere shortening is a key indicator of cellular aging and immune dysfunction.
• Measuring telomere length in specific immune cells is crucial for understanding immune aging.
• Existing methods often limit the integration of telomere length with detailed immune cell profiling.

Purpose of the Study:

• To develop a streamlined protocol for measuring relative telomere length (RTL) in peripheral blood mononuclear cells (PBMCs).
• To integrate RTL measurement with multi-parameter immunophenotyping using spectral flow cytometry.
• To provide a reproducible workflow for studying immune aging and disease-associated changes.

Main Methods:

• A novel flow cytometry-based fluorescence in situ hybridization (flow-FISH) protocol was developed.
• An 18-color spectral flow cytometry panel was designed to measure RTL and immunophenotype simultaneously.
• Optimizations included dual fixation, use of an internal control cell line (1301), and cell cycle exclusion for accurate quantification.

Main Results:

• The protocol successfully measured RTL across 15 distinct immune subsets within a single sample.
• The method demonstrated the ability to detect expected telomere shortening associated with T-cell differentiation and senescence.
• Validation in PBMCs from older adults confirmed the method's utility for immune aging studies.

Conclusions:

• The developed flow-FISH protocol offers a powerful and integrated approach for analyzing telomere length and immune cell characteristics.
• This method overcomes limitations of conventional techniques, enabling deeper insights into immune aging and age-related diseases.
• The streamlined workflow enhances reproducibility and applicability in diverse immunological research settings.