Rapid flow cytometric method for measuring senescence associated β-galactosidase activity in human fibroblasts

Gerard Noppe, Pim Dekker*, Corine De Koning-Treurniet, Joke Blom, Diana Van Heemst, Roeland W. Dirks, Hans J. Tanke, Rudi G.J. Westendorp, Andrea B. Maier

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

Senescence associated-β-galactosidase (SA-β-gal) activity is a widely used marker for cellular senenescence. SA-β-gal activity is routinely detected cytochemically, manually discriminating negative from positive cells. This method is time-consuming, subjective and therefore prone to operator-error. We aimed to optimize a flow cytometric method described by other workers using endothelial cells to better differentiate between populations of fibroblasts in degrees of SA-β-gal activity. Skin fibroblasts were isolated from young (mean age ± SD: 25.5 ± 1.8) and very old (age 90.2 ± 0.3) subjects. Different pH modulators were tested for toxicity. To induce stress-induced senescence, fibroblasts were exposed to rotenone. Senescence was assessed measuring SA-β-gal activity by cytochemistry (X-gal) and by flow cytometry (C12FDG). The pH modulator Bafilomycin A1 (Baf A1) was found to be least toxic for fibroblasts and to differentiate best between nonstressed and stressed fibroblast populations. Under nonstressed conditions, fibroblasts from very old subjects showed higher SA-β-gal activity than fibroblasts from young subjects. This difference was found for both the flow cytometric and cytochemical methods (P = 0.013 and P = 0.056 respectively). Under stress-induced conditions the flow cytometric method but not the cytochemical method revealed significant higher SA-β-gal activity in fibroblasts from very old compared to young subjects (P = 0.004 and P = 0.635 respectively). We found the modified flow cytometric method measuring SA-β-gal activity superior in discriminating between degrees of senescence in different populations of fibroblasts.

Original languageEnglish
Pages (from-to)910-916
Number of pages7
JournalCytometry Part A
Volume75
Issue number11
DOIs
Publication statusPublished - Nov 2009

Keywords

  • Flow cytometry
  • Sa-β-galactosidase
  • Senescence
  • Skin fibroblasts

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