Abstract Telomere shortening is a hallmark of aging and immune dysfunction, offering a reliable indicator of cellular replicative history and biological age. Measuring telomere length in defined immune subsets provides critical insights into immune aging and disease‐associated remodeling. However, conventional methods often require prior cell sorting or compromise surface marker detection, limiting their ability to integrate telomere length with immunophenotyping. The advent of spectral flow cytometry enables highly multiplexed single‐sample analysis, overcoming many of these technical constraints. Here, we present a streamlined protocol that combines flow cytometry–based fluorescence in situ hybridization (flow‐FISH) with spectral cytometry to measure relative telomere length (RTL) in peripheral blood mononuclear cells (PBMCs). This 18‐color panel simultaneously captures RTL across 15 immune subsets, alongside five functional and phenotypic markers reflecting activation, differentiation, senescence, and exhaustion. Our flow‐FISH method integrates telomere analysis and immune profiling into a single, reproducible workflow. Key optimizations include dual fixation to preserve membrane staining, use of the 1301 cell line as an internal telomere reference, and exclusion of dividing cells via cell cycle staining to ensure accurate RTL quantification. Validation in PBMCs from older adults demonstrated the method's ability to detect expected RTL decreases with T‐cell differentiation and senescence progression, confirming its utility for studies on immune aging, chronic infection, and other age‐related conditions. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : Staining and flow cytometry for flow‐FISH to measure telomere length and immunophenotype in peripheral blood mononuclear cells (PBMCs) Support Protocol : Preparation and cryopreservation of 1301 internal control cell line Basic Protocol 2 : Data analysis and interpretation for telomere length and immunophenotype measurements in PBMCs
Trigueros et al. (Wed,) studied this question.