Cellular Senescence Flow Cytometry Assay

Protocol tips

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	Add 10 µL of 200X SA-ß-Gal Substrate Solution and incubate at 37ºC for 4 hrs to overnight.

Protocol tips
Add 10 µL of 200X SA-ß-Gal Substrate Solution and incubate at 37ºC for 4 hrs to overnight.

Publication protocol

MSCs in P2 and P3 were seeded onto chamber slides (LAB-TEK, Nalge Nunc International, Naperville, IL) at a density of 5*103 cells/cm2 and cultured until 80–90% confluency. Cells were washed in PBS and fixed in 0.5% glutaraldehyde for 10 min. MSCs were incubated for 4 h at 37°C with fresh staining solution containing 0.1% x-Gal-solution (5-bromo-4-chloro-3-indolyl β-galactosidase; Sigma, Germany), 40 mM citric acid/sodium-phosphate pH 6, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl and 2 mM MgCl2. The cells were then counterstained with eosin and mounted with Roti-Histokitt (Carl Roth, Karlsruhe, Germany).

Quantification of SA-β-gal activity was performed using a commercial fluorometric cellular senescence assay kit (Cell Biolabs inc., San Diego, CA, USA). MSCs from healthy and CKDsev-RK donors in Passage 3 were harvested using the lysis buffer provided in the kit, and the assay was performed according to the manufacturer's instructions.

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