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Senescence Detection Using Reflected Light in Adipose Stromal Vascular Fraction.

Leo J S Westerberg¹, Laurent Barbe¹, Marcus Ehrström²

¹Department of Cell and Molecular Biology, Karolinska Institutet.

Journal of Visualized Experiments : Jove

|June 22, 2026

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We developed a new microscopy method to precisely quantify cellular senescence markers in human cells. This technique improves upon traditional assays for studying aging and metabolic diseases.

Area of Science:

Cellular and Molecular Biology
Aging Research
Biomedical Imaging

Background:

Cellular senescence, a state of permanent cell-cycle arrest, is linked to aging and metabolic diseases.
Senescence-associated β-galactosidase (SA-β-gal) activity is a key marker, but current assays lack quantitative precision, especially in complex cell mixtures like human stromal vascular fraction (SVF).
Limitations of conventional SA-β-gal assays include subjective assessment and poor quantification in heterogeneous primary human cells.

Purpose of the Study:

To present an optimized reflected light confocal microscopy protocol for high-resolution, quantitative SA-β-gal detection in human SVF cells.
To enable objective, single-cell analysis of SA-β-gal activity in heterogeneous cell populations.
To provide a more sensitive, reproducible, and quantitative alternative to conventional SA-β-gal staining methods.

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Main Methods:

Developed an optimized reflected light confocal microscopy approach.
Enabled simultaneous immunocytochemistry for multiplexed marker detection.
Incorporated pH-matched controls for accurate SA-β-gal activity measurement.

Main Results:

Achieved high-resolution, quantitative detection of SA-β-gal activity in human SVF cells.
Enabled objective single-cell analysis and simultaneous detection of other markers.
Demonstrated a sensitive, reproducible, and quantitative method for studying cellular senescence.

Conclusions:

The optimized confocal microscopy method offers a significant improvement for studying cellular senescence in heterogeneous primary human cell populations.
This approach provides objective, quantitative data, overcoming limitations of traditional SA-β-gal assays.
The protocol facilitates deeper understanding of senescence's role in aging and metabolic diseases.