Detecting Senescence in T Cells by Flow Cytometry Using the SA-β-Galactosidase Assay

Area of Science:

• Cellular biology
• Immunology
• Gerontology

Background:

• Cellular senescence is irreversible growth arrest, marked by SA-β-gal activity at pH 6.
• Traditional X-Gal substrate methods for SA-β-gal detection lack sensitivity and require fixed cells.

Purpose of the Study:

• To introduce and validate a refined method for detecting senescence-associated β-galactosidase (SA-β-gal).
• To quantify SA-β-gal in live and fixed cells using the SPiDER β-gal substrate.
• To investigate age-related changes in senescent immune cells.

Main Methods:

• Utilized the SPiDER β-gal substrate from Dojindo's Cellular Senescence Detection Kit.
• Employed flow cytometry for SA-β-gal quantification in peripheral blood mononuclear cells (PBMCs) and UVB-treated fibroblasts.
• Analyzed PBMCs from healthy donors of varying ages.

Main Results:

• The refined method demonstrated improved cell permeability and quantitative analysis capabilities.
• A progressive increase in SA-β-gal-positive CD8+ T cells was observed with advancing age.
• UVB-treated fibroblasts, a senescence model, were successfully analyzed using the new method.

Conclusions:

• The SPiDER β-gal substrate offers a sensitive and versatile tool for SA-β-gal detection in diverse cell types.
• Accumulation of senescent CD8+ T cells is a characteristic feature of aging.
• This method has broad applications in aging, immune function, and cancer biology research.