Detection and Characterization of Senescent Cells with Flow Cytometry

Cellular senescence, a stable cell cycle arrest induced by various stressors or DNA damage, plays a critical role in various aspects of human physiology, including embryonic development, wound healing, and age-related diseases like cancer. Despite its discovery nearly 50 years ago, a universal marker for senescent cells remains elusive. Current identification methods, such as the detection of senescence-associated β-galactosidase activity (SA-β-Gal), immunohistochemistry, and qPCR, have limitations, including subjective assessment of the results and complex prerequisites. Flow cytometry (FC) has emerged as a reliable technique for detecting senescent phenotypes at single-cell level, traditionally based on proteins like Ki-67 and p16INK4a and, more recently, via the detection of lipofuscin with a novel Sudan Black-B analog, namely, GLF16. Here, we present detailed protocols using GLF16 in FC, to detect senescent cells and specifically, of the human myeloma cell lines NCI-H929 and L363. In the protocols, we describe the induction of cellular senescence using H₂O₂, the staining process, and the gating strategy to accurately identify senescent cells. Additionally, we provide a comprehensive method for detecting senescent cells in bone marrow clinical samples from patients with multiple myeloma, following red blood cell lysis and leukocyte staining. These techniques broaden the potential of FC applications in the field of senescence and could be utilized as surrogate tests in clinical diagnostics.