How do you detect senescence in a cell?
The method to detect SA-beta-gal is a convenient, single cell-based assay, which can identify senescent cells even in heterogeneous cell populations and aging tissues, such as skin biopsies from older individuals. Because it is easy to detect, SA-beta-gal is currently a widely used biomarker of senescence.
How do you induce senescence in cells?
Various oxidative stresses have been used to induce premature senescence, including exposure to hydrogen peroxide (26), ultraviolet (UV) light (27), tert-butylhydroperoxide (28), and hyperoxia (18), among which hydrogen peroxide is the most commonly used inducer.
How does LacZ staining work?
The bacterial enzyme β-galactosidase (gene LacZ) is frequently used as reporter gene. It can be easily located with a LacZ stain using the artificial substrate X-gal, which turns blue when it is cleaved by β-galactosidase. This protocol describes the staining of cultured cells containing active LacZ genes.
How do you isolate senescent cells?
Technologies like tangential flow filtration can be used to isolate senescent cells. Other methods, like senolytic viruses, could be used to remove senescent cells.
How does p16 induce senescence?
The p16 mediated senescence acts through the retinoblastoma (Rb) pathway inhibiting the action of the cyclin dependant kinases leading to G1 cell cycle arrest. Rb is maintained in a hypophosphorylated state resulting in the inhibition of transcription factor E2F1.
How do you analyze cellular senescence markers?
To analyze cellular senescence markers, plate the cells in 6 cm dishes and analyze both the protein and the RNA simultaneously (see Section 7 for RNA isolation instructions). Remove the cell culture medium. Put the cells on a tray of ice. Wash the cells 2 times with cold 1x PBS.
What is the senescence β-galactosidase staining kit?
The Senescence β-Galactosidase Staining Kit is designed to conveniently provide reagents needed to detect β-galactosidase activity at pH 6, a known characteristic of senescent cells. Papers have published using this kit in both cells and frozen tissue.
What is the senescence assay?
Senescence Associated β-galactosidase Staining (Senescence assay) Principle. The identification of senescent cells is based on an increased level of lysosomal β-galactosidase activity. Cells under normal growth condition produce acid lysosomal β- galactosidase, which is localized in the lysosomes.
Can senescent phenotypes be assessed in vivo?
These protocols can be widely used to assess senescent phenotypes both in vitroand in vivo, although many caveats exist for characterizing senescence in vivo20. Other cells may express senescent markers, even if they are not senescent cells. For example, cells such as osteoclasts and macrophages have higher levels of β-gal activity32,33.