Flow Cytometry-Based Quantification of Therapy-Induced Senescent Cancer Cells

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.

First, adjust the lysosomal pH of cultured cell samples by adding a solution of one micromolar bafilomycin A in DMEM to the cell pellet at a concentration of one times 10 to the sixth cells per milliliter, and pipette to mix. Incubate for 30 minutes at 37 degrees Celsius on a rotator at a slow speed. Then, to stain for senescence-associated beta-galactosidase, add DDAOG stock solution at 10 micrograms per milliliter to the samples without washing.

Place on a rotator for 60 minutes, protected from direct light. As before, centrifuge the tubes. Remove the supernatant, and wash the pellet with one milliliter of ice-cold 0.5% BSA in PBS. Then, add 300 microliters of diluted calcine violet 450 AM solution to the washed cell pellets. Incubate for 15 minutes on ice in the dark.

Transfer the cell samples to flow cytometry instrument-compatible tubes. In the data acquisition software, open a violet channel histogram and a far-red channel versus green channel dot dot-plot. Initiate cytometer data acquisition at a low intake speed and place the positive control samples stained with DDAOG on the intake port. Begin to acquire sample data. Adjust the channel voltages such that more than 90% of events are contained within each plot.

When all settings are optimal, record 10,000 events per sample. Place the vehicle-only control samples stained with DDAOG on the intake port. Initiate data acquisition and record 10,000 events per sample. Look for an increase in autofluorescence or AF and DDAO galactoside signal versus positive control. Save the sample data in .FCS file format and export the files to a workstation computer equipped with flow cytometry analysis software.