Encyclopedia of Experiments: Immunology
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Transcript
Take cancer cells pre-treated with a chemotherapy drug that induces senescence in a percentage of cells.
These cells accumulate autofluorescent lipofuscin granules, a known senescence marker.
Add an antibiotic that enters the cells and inhibits the lysosomal proton pumps, preventing lysosomal acidification.
An increase in pH inactivates the lysosomal enzymes.
Senescent cells overexpress lysosomal β-galactosidase even at near-neutral pH, making it a senescence biomarker.
Introduce a β-galactosidase substrate. Incubate.
β-galactosidase hydrolyzes the internalized substrate into a fluorescent product, labeling the senescent cells.
Centrifuge and discard the supernatant.
Add a cell viability dye. Within live cells, active esterases convert the dye into a membrane-impermeable fluorescent product. Dead cells with compromised membrane integrity remain unstained.
Using flow cytometry, detect the viable cells displaying the dual markers lipofuscin and fluorescent β-galactosidase substrate, indicating senescent cells.
Quantify the percentage of senescent cells to demonstrate the drug efficacy.
