Rapid flow cytometric method for measuring senescence associated beta-galactosidase activity in human fibroblasts.
Senescence associated-beta-galactosidase (SA-beta-gal) activity is a widely used marker for cellular senenescence. SA-beta-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-beta-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-beta-gal activity by cytochemistry (X-gal) and by flow cytometry (C(12)FDG). 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-beta-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-beta-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-beta-gal activity superior in discriminating between degrees of senescence in different populations of fibroblasts.
Department of Gerontology and Geriatrics, Leiden University Medical Center, Leiden 2300 RC, The Netherlands.
Gerard
G
Noppe
Pim
P
Dekker
Corine
C
de Koning-Treurniet
Joke
J
Blom
Diana
D
van Heemst
Roeland W
RW
Dirks
Hans J
HJ
Tanke
Rudi G J
RG
Westendorp
Andrea B
AB
Maier
D000328:Adult; D000367:Age Factors; D000369:Aged, 80 and over; D000375:Aging; D016922:Cellular Senescence; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry / Q000379:methods; D008247:Lysosomes / Q000378:metabolism; D018942:Macrolides / Q000494:pharmacology; D008954:Models, Biological; D012402:Rotenone / Q000494:pharmacology; D012867:Skin / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism*
D004791:Enzyme Inhibitors; D018942:Macrolides; D012402:Rotenone; C040929:bafilomycin A1; D001616:beta-Galactosidase