Please use this identifier to cite or link to this item:
標題: A fluorimetric method using fluorescein di-beta-D-galactopyranoside for quantifying the senescence- associated beta-galactosidase activity in human foreskin fibroblast Hs68 cells
作者: 胡淼琳
Yang, N.C.
Hu, M.L.
關鍵字: senescence-associated beta-galactosidase activity;cell aging;X-Gal;fluorescein-di-beta-D-galactopyranoside
Project: Analytical Biochemistry
期刊/報告no:: Analytical Biochemistry, Volume 325, Issue 2, Page(s) 337-343.
The senescence-associated P-galactosidase (SA-PG) assay is one of the few accepted markers of cell aging. However, the cytochemical method using 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-Gal) as substrate is limited in sensitivity and is only semiquantitative. Here, we modified the X-Gal method by replacing X-Gal with fluorescein di-beta-D-galactopyranoside (FDG) as substrate for SA-PG, and the activity was measured fluorimetrically. We showed in Hs68 cells that the FDG fluorescein fluorescence increased with increasing passages of the cells in parallel with the X-Gal method. A major advantage of the FDG method is that it is a quantitative method for the SA-PG activity. For example, we showed that the FDG fluorescein in p30(+1) of Hs68 cells was generally stronger than that in p26(+1) cells, whereas the X-Gal method gave similar results (95 and 100%) for p26(+1) and p30(+1) cells. The FDG method was precise with a relative standard deviation lower than 10%. We further demonstrated that FDG and X-Gal could be added simultaneously for SA-PG assay because the FDG fluorescein diffused readily through formaldehyde-fixed cell membrane and could be detected in the suspension buffer. Thus, a double-substrate method, i.e., X-Gal for rapid qualitative assay and FDG for quantitative assay, can be conducted simultaneously to provide a simple and reliable assay of SA-PG activity as a marker of cell aging. (C) 2003 Elsevier Inc. All rights reserved.
ISSN: 0003-2697
DOI: 10.1016/j.ab.2003.11.012
Appears in Collections:食品暨應用生物科技學系

Show full item record

Google ScholarTM




Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.