Far-Red Fluorescent Senescence-Associated β-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry

Our multi-parameter flow cytometry protocol enables rapid and improved detection of therapy-induced senescence in tumor cells, which is a condition of ongoing study in cancer biology and therapy. Our flow cytometry senescence assay is more rapid than existing microscopy-based assays. We use two markers, instead of just one, to identify senescent cells, improving assay reliability.

Cells can be co-stained with fluorescent antibodies to detect additional targets of interest. Flow cytometry sorting can be used to enrich populations of senescent cells for downstream analysis. The assay can detect senescent cells in cultured cancer cell lines, or in whole tumor samples after gentle dissociation.

Before attempting this technique, it is important to be familiar with the general operating procedures of the flow cytometer being used. Please consult the text for recommended flow cytometer specifications. One day prior to senescence induction by drugs, harvest the cancer cell line culture with 0.25%trypsin EDTA.

Place the cells at 37 degrees Celsius for five minutes to activate trypsin, and detach the cell monolayer. When the monolayer has visibly detached, neutralize trypsin by adding an equal volume of complete culture medium. Transfer the cell suspension to a sterile conical tube.

Count the cells using the standard hemocytometer method, and record cells per milliliter. Plate cells at one times 10 to the third to 10 times 10 to the third cells per square centimeter in a standard six-well plastic culture plate. Incubate the plate overnight at 37 degrees Celsius with 5%carbon dioxide and humidity.

The next day, treat the cells with the senescence-inducing agent of interest, such as etoposide. Then incubate for four days to allow the onset of senescence. Examine the cells daily under a light microscope for expected morphology changes.

After the onset of senescence, harvest the cells by adding trypsin EDTA for five minutes at 37 degrees Celsius. When the cells are dissociated into suspension, neutralize trypsin with an equal volume of complete medium. Transfer each well of cell suspension into a 1.7-milliliter microcentrifuge tube.

Count the cells again and aliquot an equal number of cells per sample into a new set of tubes. Centrifuge the tubes for five minutes at 1, 000 g at four degrees Celsius and remove the supernatant. 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 Calcein 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 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. Using cytometry data analysis software, load the fcs data files for all samples acquired. First, to gate viable cells, double-click on the sample data for the vehicle-only control to open its data window.

Visualize the data as a violet channel histogram. The viable cells stained, stained by CV 450, are based on their brighter fluorescence than the dead cells. Draw a gate using the Single Gate Histogram tool to include viable cells only.

Name the gate Viable. Then drag the Viable gate onto the other cell samples from the sample layout window to apply the gate uniformly. Open the Layout window.

In the Layout window, visualize all samples as violet channel histograms. Verify that viable cell gating is appropriate across samples. If not, adjust as needed.

Next, to gate senescent cells, double-click on the gated viable cell data for the vehicle-only control to open its data window. Then visualize the data as a dot plot for the far-red channel versus green channel. Draw a gate using the Rectangle Gating tool to include less than 5%of DDAO-positive and AF-positive cells from the upper right quadrant.

Name the gate as Senescent. Then drag the Senescent gate onto the viable subset of all samples from the sample Layout window to apply the gate uniformly. Into the Layout window, drag and drop all viable cell-gated subsets.

Visualize all viable samples as far-red versus green channel dot plots, and observe the results. Ensure that the senescent gate is visible on all plots and that the gate for the vehicle-only controls exhibits less than 5%senescent cells. The analysis is now complete.

Data can now be exported as desired in graphical and table format. Compared to untreated cells, senescent B16-F10 melanoma cells induced by etoposide exhibited enlarged morphology and blue staining due to cleavage of X-gal by elevated senescence-associated beta-galactosidase. Staining of etoposide-treated cells with fluorescent C12FDG, or DDAOG demonstrated comparable staining patterns and intensity variations to X-gal.

However, cellular autofluorescence overlapping with green C12FDG emission was shown to accumulate in unstained senescent cells. In contrast, autofluorescence was typically negligible in the far-red emission range of DDAOG. Flow cytometer data acquisition setup for scatter plots, five-peak commercial rainbow fluorescent calibration microspheres, and single-channel fluorescence data from stained cells are shown here.

DDAOG flow cytometer senescence assay data for B16-F10 melanoma cells showed that Therapy-Induced Senescence, or TIS, induced by etoposide occurred in 35%of viable cells, and the senolytic agent ABT-263 almost eliminated TIS cells. In A549 lung cancer cells, bleomycin-induced TIS in 66%of viable cells and ABT-263 reduced the percentage to 15. ABT-263 alone was not toxic to untreated proliferating cells.

In an antibody co-staining assay, a histogram of PE channel data showed that 42%of etoposide-treated cells were positive for the senescence marker DPPP4-positive. Further, visualization with two-dimensional dot plots indicated that 44%of etoposide-treated cells were double positive for DDAOG and DPP4 versus 4%of vehicle-only cells. Fixation of DDAOG stained cells was next evaluated.

Compared to unfixed control samples, fixed samples exhibited a slightly higher background in untreated cells with a higher percentage of cells scoring as senescent in BLM-treated cells. This effect was also observed in fixed samples stored overnight, and for one week at four degrees Celsius. Flow cytometry sorting and validation of enriched senescent cell populations by morphology and proliferation markers are shown.

Sorted senescent cells displayed enlarged morphology, as expected, visualized by staining actin with fluorescent phalloidin. A reduction in signal for proliferation marker Ki67 was also observed by immunofluorescent staining. Finally, quantification of senescence in tumors treated with chemotherapy drugs was assessed.

X-gal staining in tissues was relatively weak, but blue staining was evident in tumors treated with doxorubicin, or pegylated liposomal doxorubicin, particularly in tumors that also scored positive for senescence by DDAO-galactoside flow assay. As expected, saline-only tumors exhibited negligible senescence. Here, as with any live cell assay, the protocol steps should be performed efficiently, but gently.

Prolonged staining procedures, or incubations beyond the times mentioned should be avoided. If senescent cells are flow cytometrically sorted, they can be placed back into culture for immunoassays, or lysed, and processed for omics analysis, including transcriptomics, or proteomics. This technique has enabled our lab and others to identify novel features of senescent tumor cells, including quantification of DNA damage, protein expression, and changes in metabolism.