Senescence-Associated β-Galactosidase Staining

SA-β-gal staining of hMSCs was performed as previously reported (43 (link)). In brief, cells were washed twice with PBS and fixed in fixation buffer (2% formaldehyde and 0.2% glutaraldehyde) for 5 min at room temperature. Fixed cells were stained with fresh staining buffer (40 mM citric acid/Na phosphate buffer, 5 mM K₄[Fe(CN)₆], 5 mM K₃[Fe(CN)₆], 150 mM NaCl, 2 mM MgCl₂, 1 mg/ml X-gal (AMRESCO, 0428-25G)) at 37°C overnight. Images were captured with a microscope digital camera (Olympus). The numbers of SA-β-gal-positive cells were determined with ImageJ.

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Diao Z., Ji Q., Wu Z., Zhang W., Cai Y., Wang Z., Hu J., Liu Z., Wang Q., Bi S., Huang D., Ji Z., Liu G.H., Wang S., Song M, & Qu J. (2021). SIRT3 consolidates heterochromatin and counteracts senescence. Nucleic Acids Research, 49(8), 4203-4219.

Publication 2021

X-gal microscope digital camera

Buffer Camera Cells Citric acid Formaldehyde Glutaraldehyde Mgcl2 Microscope Nacl Phosphate X gal

Corresponding Organization : Capital Medical University

Other organizations : Beijing Institute of Genomics, Shanghai Center For Bioinformation Technology

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Variable analysis

independent variables

Staining protocol (SA-β-gal staining)

dependent variables

Number of SA-β-gal-positive cells

control variables

Cell type (hMSCs)
Washing with PBS
Fixation in fixation buffer (2% formaldehyde and 0.2% glutaraldehyde) for 5 min at room temperature
Staining buffer composition (40 mM citric acid/Na phosphate buffer, 5 mM K4[Fe(CN)6], 5 mM K3[Fe(CN)6], 150 mM NaCl, 2 mM MgCl2, 1 mg/ml X-gal)
Staining duration (overnight at 37°C)

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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