Name: far-red fluorescent senescence-associated &#946;-galactosidase probe for identification and enrichment of senescent tumor cells by flow cytometry
Uploaded: 2022-09-13T13:40:00
Description: Watch this Scientific Journal Video about Far-Red Fluorescent Senescence-Associated Î²-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry at JoVE.com

We thank the Cytometry and Antibody Core Facility at the University of Chicago for support on flow cytometry instrumentation. The Animal Research Center at the University of Chicago provided animal housing.

All animal work described was approved by the Institutional Animal Care and Use Committee at the University of Chicago.
1. Preparation and storage of stock solutions
NOTE: If cells will be flow-sorted, all solutions should be prepared using sterile techniques and filtered through a 0.22 &#181;m filter device.
<ol>
	<li>Prepare a stock solution of DDAO-Galactoside at 5 mg/mL in DMSO. Aliquot at 50 &#181;L per tube (or volume desired). Store at &#87...

<ol>
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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+senescence+in+cancer:+from+mechanisms+to+detection.">Cellular senescence in cancer: from mechanisms to detection.</a> Molecular Oncology. 15 (10), 2634-2671 (2021).</li><li>Hernandez-Segura, A., Nehme, J., Demaria, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cellular+senescence.">Hallmarks of cellular senescence.</a> Trends in Cell Biology. 28 (6), 436-453 (2018).</li><li>Bojko, A., Czarnecka-Herok, J., Charzynska, A., Dabrowski, M., Sikora, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+the+senescence+phenotype+of+cancer+cells+treated+with+chemotherapeutic+agents.">Diversity of the senescence phenotype of cancer cells treated with chemotherapeutic agents.</a> Cells. 8 (12), 1501 (2019).</li><li>Miku&#322;a-Pietrasik, J., Niklas, A., Uruski, P., Tykarski, A., Ksi&#261;&#380;ek, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+significance+of+therapy-induced+and+spontaneous+senescence+of+cancer+cells.">Mechanisms and significance of therapy-induced and spontaneous senescence of cancer cells.</a> Cellular and Molecular Life Sciences. 77 (2), 213-229 (2020).</li><li>Lee, B. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipofuscin:+Mechanisms+of+formation+and+increase+with+age.">Lipofuscin: Mechanisms of formation and increase with age.</a> APMIS. 106 (2), 265-276 (1998).</li><li>Georgakopoulou, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+lipofuscin+staining+as+a+novel+biomarker+to+detect+replicative+and+stress-induced+senescence.+A+method+applicable+in+cryo-preserved+and+archival+tissues.">Specific lipofuscin staining as a novel biomarker to detect replicative and stress-induced senescence. A method applicable in cryo-preserved and archival tissues.</a> Aging. 5 (1), 37-50 (2013).</li><li>Wang, B., Demaria, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+quest+to+define+and+target+cellular+senescence+in+cancer.">The quest to define and target cellular senescence in cancer.</a> Cancer Research. 81 (24), 6087-6089 (2021).</li><li>Appelbe, O. K., Zhang, Q., Pelizzari, C. A., Weichselbaum, R. R., Kron, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-guided+radiotherapy+targets+macromolecules+through+altering+the+tumor+microenvironment.">Image-guided radiotherapy targets macromolecules through altering the tumor microenvironment.</a> Molecular Pharmaceutics. 13 (10), 3457-3467 (2016).</li><li>Maciorowski, Z., Chattopadhyay, P. K., Jain, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+multicolor+flow+cytometry.">Basic multicolor flow cytometry.</a> Current Protocols in Immunology. 117, 1-38 (2017).</li><li>Fan, Y., Cheng, J., Zeng, H., Shao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Senescent+cell+depletion+through+targeting+BCL-family+proteins+and+mitochondria.">Senescent cell depletion through targeting BCL-family proteins and mitochondria.</a> Frontiers in Physiology. 11, 593630 (2020).</li><li>Kim, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+senescent+cell+surface+targetable+protein+DPP4.">Identification of senescent cell surface targetable protein DPP4.</a> Genes and Development. 31 (15), 1529-1534 (2017).</li><li>Flor, A. C., Kron, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid-derived+reactive+aldehydes+link+oxidative+stress+to+cell+senescence.">Lipid-derived reactive aldehydes link oxidative stress to cell senescence.</a> Cell Death Discovery. 7 (9), 2366 (2016).</li><li>Jochems, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cancer+SENESCopedia:+A+delineation+of+cancer+cell+senescence.">The Cancer SENESCopedia: A delineation of cancer cell senescence.</a> Cell reports. 36 (4), 109441 (2021).</li><li>Fallah, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doxorubicin+and+liposomal+doxorubicin+induce+senescence+by+enhancing+nuclear+factor+kappa+B+and+mitochondrial+membrane+potential.">Doxorubicin and liposomal doxorubicin induce senescence by enhancing nuclear factor kappa B and mitochondrial membrane potential.</a> Life Sciences. 232, 116677 (2019).</li><li>Kasper, M., Barth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bleomycin+and+its+role+in+inducing+apoptosis+and+senescence+in+lung+cells+-+modulating+effects+of+caveolin-1.">Bleomycin and its role in inducing apoptosis and senescence in lung cells - modulating effects of caveolin-1.</a> Current Cancer Drug Targets. 9 (3), 341-353 (2009).</li><li>Muthuramalingam, K., Cho, M., Kim, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+senescence+and+EMT+crosstalk+in+bleomycin-induced+pathogenesis+of+pulmonary+fibrosis-an+in+vitro+analysis.">Cellular senescence and EMT crosstalk in bleomycin-induced pathogenesis of pulmonary fibrosis-an in vitro analysis.</a> Cell Biology International. 44 (2), 477-487 (2020).</li><li>Flor, A. C., Wolfgeher, D., Wu, D., Kron, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+signature+of+enhanced+lipid+metabolism,+lipid+peroxidation+and+aldehyde+stress+in+therapy-induced+senescence.">A signature of enhanced lipid metabolism, lipid peroxidation and aldehyde stress in therapy-induced senescence.</a> Cell Death Discovery. 3, 17075 (2017).</li><li>Burd, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+tumorigenesis+and+senescence+in+vivo+with+a+p16(INK4a)-luciferase+model.">Monitoring tumorigenesis and senescence in vivo with a p16(INK4a)-luciferase model.</a> Cell. 152 (1-2), 340-351 (2013).</li><li>Liu, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+exhibiting+strong+p16+(INK4a)+promoter+activation+in+vivo+display+features+of+senescence.">Cells exhibiting strong p16 (INK4a) promoter activation in vivo display features of senescence.</a> Proceedings of the National Academy of Sciences of the United States of America. 116 (7), 2603-2611 (2019).</li><li>Wang, L., Lankhorst, L., Bernards, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+senescence+for+the+treatment+of+cancer.">Exploiting senescence for the treatment of cancer.</a> Nature Reviews Cancer. 22 (6), 340-355 (2022).</li><li>Baek, K. -. 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Over the last decade or so, flow cytometry has become a more common assay platform in cancer research due to the emerging popularity of tumor immunology, the development of lower-cost flow cytometers, and the improvement of shared instrumentation facilities at academic institutions. Multicolor assays are now standard, as most newer instruments are equipped with violet, blue-green, and red to far-red optical arrays. Thus, this DDAOG protocol is likely to be compatible with a wide array of flow cytometers. Of course, any f...

Senescent cells normally accumulate in organisms over years during normal biological aging but may also develop rapidly in tumor cells as a response to damage induced by various cancer treatments, including radiation and chemotherapy. Though no longer proliferating, therapy-induced senescent (TIS) tumor cells may contribute to treatment resistance and drive recurrence1,2,3. Factors secreted by TIS cells can exacerbate tumor malignancy by promoting immune evasion or metastasis4,5. TIS cells develop complex, context-specific phenotypes, altered metabolic profiles, and unique immune responses6,7,8. Therefore, the identification and characterization of TIS tumor cells induced by various cancer treatment approaches is a topic of ongoing interest to the cancer research community.
To detect TIS tumor cells, conventional senescence assays are widely used, primarily based on detecting increased activity of the senescence marker enzyme, the lysosomal beta-galactosidase GLB19. Detection at a near-neutral (rather than acidic) lysosomal pH allows for specific detection of senescence-associated beta-galactosidase (SA-&#946;-Gal)10. A standard SA-&#946;-Gal assay that has been used for several decades uses X-Gal (5-bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside), a blue chromogenic beta-galactosidase substrate, to detect SA-&#946;-Gal in fixed cells by light microscopy11. The X-Gal assay allows the qualitative visual confirmation of TIS utilizing commonly available reagents and laboratory equipment. A basic transmitted light microscope is the only instrumentation required to evaluate the presence of the blue chromogen. However, the X-Gal staining procedure can lack sensitivity, sometimes requiring more than 24 h for color to develop. Staining is followed by low-throughput, subjective scoring of individual senescent cells based on counting the cells exhibiting some level of intensity of the blue chromogen under a light microscope. As X-Gal is cell-impermeable, this assay requires solvent-fixed cells, which cannot be recovered for downstream analysis. When working with limited samples from animals or patients, this can be a major drawback.
Improved SA-&#946;-Gal assays using cell-permeant, fluorescent enzyme substrates, including C12-FDG (5-dodecanoylaminofluorescein Di-&#946;-D-Galactopyranoside, green) and&#160;DDAOG (9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) &#946;-D-Galactopyranoside, far-red)&#160;have previously appeared in the literature12,13,14,15. The chemical probe structure and optical characteristics of DDAOG are shown in Supplementary Figure S1. These cell-permeant probes&#160;permit the analysis of living (rather than fixed) cells, and fluorescent rather than chromogenic probes facilitate the use of rapid high-throughput fluorescent analysis platforms, including high-content screening instruments and flow cytometers. Sorting flow cytometers enable the recovery of enriched populations of living senescent cells from cell cultures or tumors for downstream analysis (e.g., western blotting, ELISA, or &#39;omics). Fluorescence analysis also provides a quantitative signal, allowing for more accurate determination of the percentage of senescent cells within a given sample. Additional fluorescent probes, including viability probes and fluorophore-labeled antibodies, can readily be added for multiplexed analysis of targets beyond SA-&#946;-Gal.
Similar to DDAOG, C12-FDG is a fluorescent probe for SA-&#946;-Gal, but its green fluorescent emission overlaps with intrinsic cellular AF, which arises during senescence due to the accumulation of lipofuscin aggregates in cells16. By utilizing the far-red DDAOG probe, green cellular AF can be used as a secondary parameter to confirm senescence17. This improves assay reliability by using a second marker in addition to SA-&#946;-Gal, which can often be unreliable as a single marker for senescence18. As the detection of endogenous AF in senescent cells is a label-free approach, it is a rapid and simple way to expand the specificity of our DDAOG-based assay.
In this protocol, we demonstrate the use of DDAOG and AF as a rapid, dual-parameter flow cytometry assay for the identification of viable TIS tumor cells from in vitro cultures or isolated from drug-treated tumors established in mice (Figure 1). The protocol uses fluorophores compatible with a wide range of standard commercial flow cytometry analyzers and sorters (Table 1). Quantitation of the percentage of viable senescent cells using standard flow cytometry analysis is enabled. If desired, an optional immunolabeling step may be performed to evaluate cell surface antigens of interest concurrently with senescence. Identified senescent cells can also be enriched using standard fluorescence-activated cell sorting (FACS) methodology.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64176/64176fig01.jpg" /> 
Figure 1: Experimental workflow. A schematic summarizing key points of the DDAOG assay. (A) A TIS-inducing drug is added to mammalian cultured cells or administered to tumor-bearing mice. Time is then allowed for the onset of TIS: for cells, 4 days following treatment; for mice, 22 days total, with three treatments every 5 days plus 7 days recovery. Cells are harvested or tumors are dissociated into suspension.&#160;(B)&#160;Samples are treated with Baf to adjust lysosomal pH for detection of SA-&#946;-Gal for 30 min; then, DDAOG probe is added for 60 min to detect SA-&#946;-Gal. Samples are washed 2x in PBS, and a viability stain is briefly added (15 min). Optionally, samples can be stained with fluorescent antibodies in open fluorescence channels and/or fixed for later analysis. (C) Samples are analyzed using a standard flow cytometer. Viable cells are visualized in dot plots showing red DDAOG (indicating SA-&#946;-Gal) versus green autofluorescence (lipofuscin). A gate to determine the percentage of TIS cells is established based on untreated control samples (not shown). If a sorting cytometer (FACS) is used, TIS cells can be collected and placed back into culture for further in vitro assays or lysed and processed for molecular biology assays. Abbreviations: DDAO = 9H-(1,3-dichloro-9,9-dimethylacridin-2-one); DDAOG = DDAO-Galactoside; TIS = therapy-induced senescence; FL-Ab = fluorophore-conjugated antibody; Baf = Bafilomycin A1; SA-&#946;-Gal = senescence-associated beta-galactosidase; PBS = phosphate-buffered saline; FACS = fluorescence-activated cell sorting. <a href="https://www.jove.com/files/ftp_upload/64176/64176fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fluorophore</td>
			<td>Detects</td>
			<td>Ex/Em (nm)</td>
			<td>Cytometer laser (nm)</td>
			<td>Cytometer detector / bandpass filter (nm)</td>
		</tr>
		<tr>
			<td>DDAOG</td>
			<td>SA-&#946;-Gal</td>
			<td>645/6601</td>
			<td>640</td>
			<td>670 / 30</td>
		</tr>
		<tr>
			<td>AF</td>
			<td>Lipofuscin</td>
			<td>&#60; 600</td>
			<td>488</td>
			<td>525 / 50</td>
		</tr>
		<tr>
			<td>CV450</td>
			<td>Viability</td>
			<td>408/450</td>
			<td>405</td>
			<td>450 / 50</td>
		</tr>
		<tr>
			<td>PE</td>
			<td>Antibody/surface marker</td>
			<td>565/578</td>
			<td>561</td>
			<td>582 / 15</td>
		</tr>
	</tbody>
</table>
Table 1: Fluorophores and cytometer optical specifications. Cytometer specifications used in this protocol are listed for an instrument with a total of 4 lasers and 15 emission detectors.&#160;DDAOG detected at 645/660 nm is the form of the probe cleaved by SA-&#946;-Gal1. Uncleaved DDAOG can exhibit low level fluorescence at 460/610 nm but is removed by wash steps in the protocol. Abbreviations: DDAO = 9H-(1,3-dichloro-9,9-dimethylacridin-2-one); DDAOG = DDAO-Galactoside; AF = autofluorescence; PE = phycoerythrin; SA-&#946;-Gal = senescence-associated beta-galactosidase.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bafilomycin A1</td>
			<td>Research Products International</td>
			<td>B40500</td>
			<td></td>
		</tr>
		<tr>
			<td>Bleomycin sulfate&#160;</td>
			<td>Cayman</td>
			<td>13877</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>US Biological</td>
			<td>A1380</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein Violet 450 AM viability dye</td>
			<td>ThermoFisher Scientific</td>
			<td>65-0854-39</td>
			<td>eBioscience</td>
		</tr>
		<tr>
			<td>DPP4 antibody, PE conjugate</td>
			<td>Biolegend</td>
			<td>137803</td>
			<td>Clone H194-112</td>
		</tr>
		<tr>
			<td>Cell line: A549 human lung adenocarcinoma</td>
			<td>American Type Culture Collection</td>
			<td>CCL-185</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell line: B16-F10 mouse melanoma</td>
			<td>American Type Culture Collection</td>
			<td>CRL-6475</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Corning</td>
			<td>3008</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainers, 100 &#181;m</td>
			<td>Falcon</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>DDAO-Galactoside</td>
			<td>Life Technologies</td>
			<td>D6488</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM medium 1x</td>
			<td>Life Technologies</td>
			<td>11960-069</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D2438</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>Sigma</td>
			<td>DN25</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxorubicin, hydrochloride injection (USP)</td>
			<td>Pfizer</td>
			<td>NDC 0069-3032-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxorubicin, PEGylated liposomal (USP)</td>
			<td>Sun Pharmaceutical</td>
			<td>NDC 47335-049-40</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA 0.5 M</td>
			<td>Life Technologies</td>
			<td>15575-038</td>
			<td></td>
		</tr>
		<tr>
			<td>Etoposide&#160;</td>
			<td>Cayman</td>
			<td>12092</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Omega&#160;</td>
			<td>FB-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc receptor blocking reagent</td>
			<td>Biolegend</td>
			<td>101320</td>
			<td>Anti-mouse CD16/32</td>
		</tr>
		<tr>
			<td>Flow cytometer (cell analyzer)</td>
			<td>Becton Dickinson (BD)</td>
			<td>Various</td>
			<td>LSRFortessa</td>
		</tr>
		<tr>
			<td>Flow cytometer (cell sorter)</td>
			<td>Becton Dickinson (BD)</td>
			<td>Various</td>
			<td>FACSAria</td>
		</tr>
		<tr>
			<td>GlutaMax 100x</td>
			<td>Life Technologies</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES 1 M</td>
			<td>Lonza</td>
			<td>BW17737</td>
			<td></td>
		</tr>
		<tr>
			<td>Liberase TL</td>
			<td>Sigma</td>
			<td>5401020001</td>
			<td>Roche</td>
		</tr>
		<tr>
			<td>Paraformaldehyde 16%</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin 100x</td>
			<td>Life Technologies</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS) 1x</td>
			<td>Corning</td>
			<td>MT21031CV</td>
			<td>Dulbecco&#39;s PBS (without calcium and magnesium)</td>
		</tr>
		<tr>
			<td>Rainbow calibration particles, ultra kit</td>
			<td>SpheroTech</td>
			<td>UCRP-38-2K</td>
			<td>3.5-3.9 &#181;m, 2E6/mL</td>
		</tr>
		<tr>
			<td>RPMI-1640 medium 1x</td>
			<td>Life Technologies</td>
			<td>11875-119</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride 0.9% (USP)</td>
			<td>Baxter Healthcare Corporation</td>
			<td>2B1324</td>
			<td></td>
		</tr>
		<tr>
			<td>Software for cytometer data acquisition, &#34;FACSDiva&#34;</td>
			<td>Becton Dickinson (BD)</td>
			<td>n/a</td>
			<td>Contact BD for license</td>
		</tr>
		<tr>
			<td>Software for cytometer data analysis, &#34;FlowJo&#34;</td>
			<td>TreeStar</td>
			<td>n/a</td>
			<td>Contact TreeStar for license</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.25%</td>
			<td>Life Technologies</td>
			<td>25200-114</td>
			<td></td>
		</tr>
	</tbody>
</table>

far-red fluorescent senescence-associated &#946;-galactosidase probe for identification and enrichment of senescent tumor cells by flow cytometry

Cellular senescence is a state of proliferative arrest induced by biological damage that normally accrues over years in aging cells but may also emerge rapidly in tumor cells as a response to damage induced by various cancer treatments. Tumor cell senescence is generally considered undesirable, as senescent cells become resistant to death and block tumor remission while exacerbating tumor malignancy and treatment resistance. Therefore, the identification of senescent tumor cells is of ongoing interest to the cancer research community. Various senescence assays exist, many based on the activity of the well-known senescence marker, senescence-associated beta-galactosidase (SA-&#946;-Gal). 
Typically, the SA-&#946;-Gal assay is performed using a chromogenic substrate (X-Gal) on fixed cells, with the slow and subjective enumeration of "blue" senescent cells by light microscopy. Improved assays using cell-permeant, fluorescent SA-&#946;-Gal substrates, including C12-FDG (green) and DDAO-Galactoside (DDAOG; far-red), have enabled the analysis of living cells and allowed the use of high-throughput fluorescent analysis platforms, including flow cytometers. C12-FDG is a well-documented probe for SA-&#946;-Gal, but its green fluorescent emission overlaps with intrinsic cellular autofluorescence (AF) that arises during senescence due to the accumulation of lipofuscin aggregates. By utilizing the far-red SA-&#946;-Gal probe DDAOG, green cellular autofluorescence can be used as a secondary parameter to confirm senescence, adding reliability to the assay. The remaining fluorescence channels can be used for cell viability staining or optional fluorescent immunolabeling.
Using flow cytometry, we demonstrate the use of DDAOG and lipofuscin autofluorescence as a dual-parameter assay for the identification of senescent tumor cells. Quantitation of the percentage of viable senescent cells is performed. If desired, an optional immunolabeling step may be included to evaluate cell surface antigens of interest. Identified senescent cells can also be flow cytometrically sorted and collected for downstream analysis. Collected senescent cells can be immediately lysed (e.g., for immunoassays or 'omics analysis) or further cultured.

Several experiments were performed to demonstrate the comparability of DDAOG to X-Gal and C12-FDG for the detection of senescence by SA-&#946;-Gal. First, X-Gal was used to stain senescent B16-F10 melanoma cells induced by ETO (Figure 2A). An intense blue color developed in a subset of ETO-treated cells, while other cells exhibited less intense blue staining. Morphology was enlarged in most ETO-treated cells. Staining ETO-treated cells with fluorescent SA-&#946;-Gal substrate C<su...

Watch this Scientific Journal Video about Far-Red Fluorescent Senescence-Associated Î²-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry at JoVE.com

Far-Red Fluorescent Senescence-Associated &#946;-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry

A protocol for fluorescent, flow cytometric quantification of senescent cancer cells induced by chemotherapy drugs in cell culture or in murine tumor models is presented. Optional procedures include co-immunostaining, sample fixation to facilitate large batch or time point analysis, and the enrichment of viable senescent cells by flow cytometric sorting.

Cellular senescence is a state of proliferative arrest induced by biological damage that normally accrues over years in aging cells but may also emerge ...

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.

Research

JoVE Journal

Cancer Research

Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median ...

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery

Aberrant cell signaling plays a central role in cancer development and progression. Most novel targeted therapies are indeed directed at proteins and ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Cell Cycle-specific Measurement of &#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer ...

Automated Dissection Protocol for Tumor Enrichment in Low Tumor Content Tissues

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly ...

Fluorescence Microscopy for ATP Internalization Mediated by Macropinocytosis in Human Tumor Cells and Tumor-xenografted Mice

Adenosine triphosphate (ATP), including extracellular ATP (eATP), has been shown to play significant roles in various aspects of tumorigenesis, such as ...

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated ...

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...