Name: Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells (Video) | JoVE Journal
Uploaded: 2022-07-12T14:30:00.000+00:00
Duration: 8 min 56 s
Description: Learn imaging flow cytometry for detecting multiple senescence markers in cancer cells using DLBCL cell lines. This method enables rapid high-resolution analysis of fluorescent signals with spatial distribution data.

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5 minutes

This work was supported by a grant to Yong Yu from Johannes Kepler University Linz (BERM16108001).

1. DLBCL cell lines with mafosfamide or daunorubicin treatment to induce cellular senescence
NOTE: The protocol also works for adherent cancer cells. Depending on cell size, seed 1-2 &#215; 105 cells into one well of a 6-well plate and incubate the plate in a 5% CO2, 37 &#176;C incubator overnight before treatment. The protocol steps are the same as for suspension cells but with two exceptions. First, cells need to be trypsinized off the plate after step 3.4. Second, wash steps are performed without centrifugation before trypsinization.
<ol>
	<li>Count DLBCL cells and seed 1 &#215; 106 cells/mL in 4 mL of medium per well into a 6-well culture plate. Cultivate DLBCL cells in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin.</li>
	<li>Add MAF (5 &#181;g/mL) or DN (20 ng/mL) into the cell culture and gently rock the plate to mix. 
	NOTE: MAF and DN are not stable. After dissolving in DMSO, the stock solution should be aliquoted and stored at -20 &#176;C. Freeze/thaw cycles should be avoided.</li>
	<li>Incubate the plate in a 5% CO2, 37 &#176;C incubator for 3 days.</li>
	<li>After a 3 day incubation, collect the DLBCL cells in 15 mL sterile centrifuge tubes and spin at 100 &#215; g, 4 &#176;C for 5 min.</li>
	<li>Discard the supernatant, resuspend the cell pellets with 4 mL of fresh medium, and add the suspensions back into the 6-well plate.</li>
	<li>Cultivate the cell plate in a 5% CO2, 37 &#176;C incubator for another 2 days before harvesting for analysis.</li>
</ol>
2. Prepare solutions for staining (Table 1)
3. Stain DLBCL cells with different senescence markers
NOTE: Cell samples stained with individual markers (i.e., pacific blue-EdU, C12FDG, or Alexa Fluor 647-&#947;H2AX) are prepared to generate a compensation matrix to correct the fluorescence spillover during measurement. Although highly suggested, this step could be suspended when there is an omittable overlap (compensation coefficient value &#8804; 0.1) of the emission spectra among different fluorophores. However, users must determine standardized compensation steps when using different instruments and fluorescent panels.
<ol>
	<li>Add 10 mM EdU solution at a ratio of 1:1,000 into the DLBCL cell culture generated from step 1.6 (final EdU concentration is 10 &#181;M). Gently rock the plate to mix and incubate in a 5% CO2, 37 &#176;C incubator for 3 h.</li>
	<li>Take the plate out of the incubator and add 100 mM chloroquine solution to a final concentration of 75 &#181;M (see discussion). Gently rock the plate to mix and incubate in a 5% CO2, 37 &#176;C incubator for 30 min.</li>
	<li>Take the plate out of the incubator and add 20 mM C12FDG solution at 1: 1,000 to a final C12FDG concentration of 20 &#181;M. Gently rock the plate to mix and incubate in a 5% CO2, 37 &#176;C incubator for 1 h.</li>
	<li>Take the plate out of the incubator and add 100 mM 2-phenylethyl-&#946;-D-thiogalactoside (PETG) solution at 1:50 to stop the fSA-&#946;-gal staining (final PETG concentration of 2 mM). Gently rotate the plate to mix.</li>
	<li>Transfer the cells to 15 mL sterile centrifuge tubes and spin at 100 &#215; g, 4 &#176;C for 5 min. Discard the supernatant and wash the cells with 4 mL of phosphate-buffered saline (PBS).</li>
	<li>Repeat the PBS washing step. Discard the supernatant and resuspend the cell pellets in 500 &#181;L of 4% paraformaldehyde fixation solution.</li>
	<li>After 10 min incubation at room temperature, centrifuge at 250 &#215; g, room temperature for 5 min. Discard the supernatant and wash cells with 4 mL of PBS.</li>
	<li>Repeat the PBS washing step. Discard the supernatant, resuspend the cell pellet in 200 &#181;L of saponin permeabilization buffer and transfer the suspension to a new 1.5 mL tube. After 10 min incubation at room temperature, centrifuge at 250 &#215; g, room temperature for 5 min.</li>
	<li>Discard the supernatant and resuspend the cell pellets in 200 &#181;L of primary antibody solution (1:500 &#947;H2AX antibody in antibody incubation solution). Incubate at 4 &#176;C overnight in the dark.</li>
	<li>Centrifuge the tubes at 250 &#215; g, 4 &#176;C for 5 min. Discard the supernatant and wash with 100 &#181;L of saponin wash solution.</li>
	<li>Repeat the washing step an additional two times. Discard the supernatant and resuspend the cell pellets in 500 &#181;L of EdU detection cocktail.</li>
	<li>Incubate at room temperature for 30 min in the dark. Centrifuge at 250 &#215; g, room temperature for 5 min. Discard the supernatant and wash with 1 mL of saponin wash solution.</li>
	<li>Repeat the washing step two times. Discard the supernatant and resuspend the cell pellets in 20-50 &#181;L of PBS. Proceed to section 4 for sample measurement.</li>
</ol>
4. Imaging senescence markers using the imaging flow cytometer system
<ol>
	<li>Empty the waste fluid bottle. Check the levels of speed beads, sterilizer, cleaner, debubbler, sheath, and rinse reagents (deionized water) to ensure sufficient fluids before turning on the instrument (see the Table of Materials).</li>
	<li>Turn on the instrument and imaging software (see the software interface in Figure 1A).</li>
	<li>Click on the Startup button to initialize fluidics and system calibration. 
	NOTE: This procedure takes approximately 45 min.</li>
	<li>Set the magnification to 40x, set fluidics speed to low, and turn on the lasers needed in the experiment. Turn on 405 nm, 488 nm, and 642 nm lasers to measure EdU-pacific blue, C12FDG, and Alexa Fluor 647-&#947;H2AX (or Alexa Fluor 647-Ki67), respectively. Set Ch6 for scatter channel, and Ch1 and Ch9 for brightfield.</li>
	<li>Start with the sample expected to have the highest fluorescence to set up the intensity of the lasers. Gently flip the sample tube to mix. Open the tube lid, and insert the sample tube onto the dock. Click on Load to start.</li>
	<li>Open a scatter plot and select the features Area_M01 and Aspect Ratio_M01 for the X- and Y-axes, respectively. Set a gate above aspect ratio of 0.5 to exclude doublets and cell aggregates below and on the right side, and the speed bead population on the left side (Figure 1B).</li>
	<li>Open a histogram plot and select the feature Gradient RMS_M01_Ch01 for the X-axis. Choose the singlet population and set the gate to select for focused cells (Figure 1C). 
	NOTE: The imaging system will automatically use speed beads to adjust the focus for imaging. However, it is recommended to select the right half of the histogram peak for the best-focused population.</li>
	<li>Open a histogram plot and select Raw Max Pixel Intensities for X-axis for each color channel (Ch2, 7, and 11). Adjust the laser powers (i.e., 488 nm, 405 nm, and 642 nm lasers for Ch2, 7, and 11, respectively), so each fluorochrome has a Raw Max Pixel value between 100 and 4,000 to avoid oversaturation. 
	NOTE: For this experiment, the laser power setting is 50 mW, 200 mW, and 50 mW for lasers 488 nm, 405 nm, and 642 nm, respectively.</li>
	<li>Choose the focused population to record and click on Acquire to measure the DLBCL samples with consistent settings. When changing samples for measurement, click on Return to recover the sample tube. Press Load to discard the sample.</li>
	<li>After all the samples are measured, turn off the brightfield and scatter laser. Measure single-color control samples to generate a compensation matrix.</li>
	<li>Click on Shutdown to close the imaging system.</li>
	<li>Analyze the data in the image analysis software.
	<ol>
		<li>Use the Spot Wizard tool of the image analysis software to automatically count and quantify nuclear &#947;H2AX foci in live-cell images. Select two cell populations (one with high and one with low spot count) to train the spot wizard for further automatic spot count analysis.</li>
	</ol>
	</li>
</ol>

<ol><li>Kuilman, T., Michaloglou, C., Mooi, W. J., Peeper, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+essence+of+senescence%5BTitle%5D)+AND+%22Genes+and+Development%22%5BJournal%5D)">The essence of senescence.</a> Genes and Development. 24 (22), 2463-2479 (2010).</li>
<li>Di Micco, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oncogene-induced+senescence+is+a+DNA+damage+response+triggered+by+DNA+hyper-replication%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication.</a> Nature. 444 (7119), 638-642 (2006).</li>
<li>Milanovic, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Senescence-associated+reprogramming+promotes+cancer+stemness%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Senescence-associated reprogramming promotes cancer stemness.</a> Nature. 553 (7686), 96-100 (2018).</li>
<li>Passos, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Feedback+between+p21+and+reactive+oxygen+production+is+necessary+for+cell+senescence%5BTitle%5D)+AND+%22Molecular+Systems+Biology%22%5BJournal%5D)">Feedback between p21 and reactive oxygen production is necessary for cell senescence.</a> Molecular Systems Biology. 6 (1), 347(2010).</li>
<li>Lee, S., Schmitt, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+dynamic+nature+of+senescence+in+cancer%5BTitle%5D)+AND+%22Nature+Cell+Biology%22%5BJournal%5D)">The dynamic nature of senescence in cancer.</a> Nature Cell Biology. 21 (1), 94-101 (2019).</li>
<li>Toussaint, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stress-induced+premature+senescence+or+stress-induced+senescence-like+phenotype%3A+one+in+vivo+reality%2C+two+possible+definitions%5BTitle%5D)+AND+%22The+Scientific+World+Journal%22%5BJournal%5D)">Stress-induced premature senescence or stress-induced senescence-like phenotype: one in vivo reality, two possible definitions.</a> The Scientific World Journal. 2, 230-247 (2002).</li>
<li>Collado, M., Blasco, M. A., Serrano, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+senescence+in+cancer+and+aging%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Cellular senescence in cancer and aging.</a> Cell. 130 (2), 223-233 (2007).</li>
<li>Munoz-Espin, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Programmed+cell+senescence+during+mammalian+embryonic+development%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Programmed cell senescence during mammalian embryonic development.</a> Cell. 155 (5), 1104-1118 (2013).</li>
<li>Faget, D. V., Ren, Q., Stewart, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unmasking+senescence%3A+context-dependent+effects+of+SASP+in+cancer%5BTitle%5D)+AND+%22Nature+Reviews+Cancer%22%5BJournal%5D)">Unmasking senescence: context-dependent effects of SASP in cancer.</a> Nature Reviews Cancer. 19 (8), 439-453 (2019).</li>
<li>Childs, B. G., Durik, M., Baker, D. J., van Deursen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+senescence+in+aging+and+age-related+disease%3A+from+mechanisms+to+therapy%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">Cellular senescence in aging and age-related disease: from mechanisms to therapy.</a> Nature Medicine. 21 (12), 1424-1435 (2015).</li>
<li>Chandra, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+reorganization+of+the+nuclear+landscape+in+senescent+cells%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Global reorganization of the nuclear landscape in senescent cells.</a> Cell Rep. 10 (4), 471-483 (2015).</li>
<li>Rodier, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Persistent+DNA+damage+signalling+triggers+senescence-associated+inflammatory+cytokine+secretion%5BTitle%5D)+AND+%22Nature+Cell+Biology%22%5BJournal%5D)">Persistent DNA damage signalling triggers senescence-associated inflammatory cytokine secretion.</a> Nature Cell Biology. 11 (8), 973-979 (2009).</li>
<li>Schleich, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((H3K9me3-mediated+epigenetic+regulation+of+senescence+in+mice+predicts+outcome+of+lymphoma+patients%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">H3K9me3-mediated epigenetic regulation of senescence in mice predicts outcome of lymphoma patients.</a> Nature Communications. 11 (1), 3651(2020).</li>
<li>Di Micco, R., Krizhanovsky, V., Baker, D., d'Adda di Fagagna, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+senescence+in+ageing%3A+from+mechanisms+to+therapeutic+opportunities%5BTitle%5D)+AND+%22Nature+Reviews+Molecular+Cell+Biology%22%5BJournal%5D)">Cellular senescence in ageing: from mechanisms to therapeutic opportunities.</a> Nature Reviews Molecular Cell Biology. 22 (2), 75-95 (2021).</li>
<li>Fan, D. N., Schmitt, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detecting+markers+of+therapy-induced+senescence+in+cancer+cells%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Detecting markers of therapy-induced senescence in cancer cells.</a> Methods in Molecular Biology. 1534, 41-52 (2017).</li>
<li>Schmitt, C. A., Lowe, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Apoptosis+and+chemoresistance+in+transgenic+cancer+models%5BTitle%5D)+AND+%22Journal+of+Molecular+Medicine%22%5BJournal%5D)">Apoptosis and chemoresistance in transgenic cancer models.</a> Journal of Molecular Medicine. 80 (3), 137-146 (2002).</li>
<li>Dorr, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synthetic+lethal+metabolic+targeting+of+cellular+senescence+in+cancer+therapy%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Synthetic lethal metabolic targeting of cellular senescence in cancer therapy.</a> Nature. 501 (7467), 421-425 (2013).</li>
<li>Fan, D. N. Y., Schmitt, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genotoxic+stress-induced+senescence%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Genotoxic stress-induced senescence.</a> Methods in Molecular Biology. 1896, 93-105 (2019).</li>
<li>Noppe, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+flow+cytometric+method+for+measuring+senescence+associated+beta-galactosidase+activity+in+human+fibroblasts%5BTitle%5D)+AND+%22Cytometry+A%22%5BJournal%5D)">Rapid flow cytometric method for measuring senescence associated beta-galactosidase activity in human fibroblasts.</a> Cytometry A. 75 (11), 910-916 (2009).</li>
<li>Biran, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+identification+of+senescent+cells+in+aging+and+disease%5BTitle%5D)+AND+%22Aging+Cell%22%5BJournal%5D)">Quantitative identification of senescent cells in aging and disease.</a> Aging Cell. 16 (4), 661-671 (2017).</li>
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The authors have no conflicts of interest to disclose.

This method examined the senescence-entering capability of four different DLBCL cell lines upon chemotherapy treatment, with bright-field imaging and flow cytometry-based quantification. On a single-cell level, we successfully detected major C12FDG+EdU-Ki67+ senescent populations in treated KARPAS422 and WSU-DLCL2 cells, and to a lesser extent in OCI-LY1 cells, while the SU-DHL6 cell line was resistant to the treatment. The difference in senescence-entering capability among cel...

A variety of stimuli can trigger cellular senescence, causing cells to enter a state of stable cell cycle arrest. These stimuli include intrinsic signaling changes or extrinsic stresses. Intrinsic signals include progressive telomere shortening, changes in telomere structure, epigenetic modification, proteostasis disorders, mitochondrial dysfunction, and activation of oncogenes. Extrinsic stresses include inflammatory and/or tissue damage signals, radiation or chemical treatment, and nutritional deprivation1,2,3,4. Among distinct types of senescence, the most commonly seen and well-studied are replicative senescence, oncogene-induced senescence (OIS), radiation-induced senescence, and therapy-induced senescence (TIS). OIS is an acute cellular response to genotoxic damage caused by replicative stress generated by aberrant oncogene activation and can to some extent prevent the pathological progression from a preneoplastic lesion to a full-blown tumor. TIS happens when tumor cells are stressed by chemotherapeutic drugs or ionizing radiation5,6.
Senescence is considered a double-edged sword in pathology due to its highly dynamic nature. It was initially described as a beneficial tumor-suppressive mechanism to remove damaged cells from the circulating pool of dividing cells, safeguarding the normal function of organs and inhibiting tumor growth7,8,9. However, emerging evidence has suggested a dark side of senescence. Senescent cells secrete proinflammatory cytokines, known as senescence-associated secretory phenotype (SASP), leading to fibrosis and malfunctional organs and promoting tumor initiation and progression10. Moreover, senescent cancer cells undergo epigenetic and gene-expression reprogramming in parallel with chromatin remodeling and activation of a sustained DNA-damage response (DDR)11,12, newly acquiring new cancer-stem-cell properties3. Although senescence-capable tumors respond better to therapeutic intervention compared to senescence-incapable ones13, the persistence of senescent cells may lead to a poor long-term prognosis if they are not effectively identified and eliminated by senolytic drugs5. Either way, a reliable method to assess senescence is of significant clinical interest, not only for the prognosis of therapy treatment but also for the development of novel strategies targeting senescent cells.
Regardless of different triggers, senescent cells exhibit some common features, including enlarged, flattened, multinucleated morphology with big vacuoles, significantly expanded nuclei, formation of H3K9me3-rich senescence-associated heterochromatin (SAHF) in the nucleus, persistent accumulation of DNA damage marker &#947;H2AX foci, activated p53-p21CIP1 and Rb-p16INK4a cell cycle regulatory mechanisms, stable G1 cell cycle arrest, massive induction of SASP, and elevated senescence-associated &#946;-galactosidase (SA-&#946;-gal) activity14. Since no single marker is sufficient to define senescence, enzymatic staining for SA-&#946;-gal activity, which is considered the gold standard for senescence detection, is usually combined with immunohistochemical staining for H3K9me3 and Ki67 to detect TIS15. However, chemical chromogenic-based SA-&#946;-gal is difficult to quantify. Here, we combined 5-dodecanoylaminofluorescein-di-&#946;-D-galactopyranoside (C12FDG) fluorescence-based SA-&#946;-gal (fSA-&#946;-gal) detection with immunofluorescent staining for &#947;H2AX and EdU-incorporated DNA to identify C12FDG+EdU-&#947;H2AX+ senescent cells using the advanced imaging flow cytometer system, which combines the speed, sensitivity and detailed single-cell images with spatial information that cannot be provided by flow cytometry and microscopy. This method enables rapid generation of high-resolution images allowing for the positioning and quantification of fluorescent signals within cells, while licensing the swift analysis of multiple samples by building standard pipelines.

<table><tbody><tr><td>Alexa Fluor 647 anti-H2A.X Phospho (Ser139) Antibody</td><td>Biolegend</td><td>613407</td><td></td></tr><tr><td>Anti-Ki-67 Mouse Monoclonal Antibody (Alexa Fluor 647)</td><td>Biolegend</td><td>350509</td><td></td></tr><tr><td>C12FDG (5-Dodecanoylaminofluorescein Di-&amp;beta;-D-Galactopyranoside)</td><td>Fisher Scientific</td><td>11590276</td><td></td></tr><tr><td>Chloroquin -diphosphat</td><td>Sigma aldrich</td><td>C6628</td><td></td></tr><tr><td>Cleanser (Coulter Clenz)</td><td>Beckman Coulter</td><td>8546929</td><td></td></tr><tr><td>Click-iT EdU Pacific Blue Flow Cytometry Assay Kit</td><td>Thermo Scientific</td><td>C10418</td><td></td></tr><tr><td>Daunorubicin</td><td>Medchemexpress</td><td>HY-13062A</td><td></td></tr><tr><td>Debubbler (70% Isopropanol)</td><td>Millipore</td><td>1.3704</td><td></td></tr><tr><td>Image Analysis software (Amnis&amp;nbsp;IDEAS&amp;nbsp;6.3)</td><td>Luminex</td><td>CN-SW69-12</td><td></td></tr><tr><td>Instrument and imaging software (Amnis&amp;nbsp;ImageStreamX&amp;nbsp;Mk II Imaging Flow Cytometer System and INSPIRE software)</td><td>Luminex</td><td>100220</td><td></td></tr><tr><td>KARPAS</td><td>DSMZ</td><td>ACC 31</td><td></td></tr><tr><td>mafosfamide cyclohexylamine</td><td>Niomech</td><td>D-17272</td><td></td></tr><tr><td>OCI-LY1</td><td>DSMZ</td><td>ACC 722</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Fisher Scientific</td><td>11473704</td><td></td></tr><tr><td>PETG (2-Phenylethyl-&amp;beta;-D-thiogalactosid)&amp;nbsp;</td><td>Sigma aldrich</td><td>P4902</td><td></td></tr><tr><td>saponin</td><td>Sigma aldrich</td><td>47036</td><td></td></tr><tr><td>Sheath</td><td>Millipore</td><td>BSS-1006-B</td><td></td></tr><tr><td>SpeedBead Kit for ImageStream</td><td>Luminex</td><td>400041</td><td></td></tr><tr><td>Sterilizer (0.4-0.7% Hypochlorite)</td><td>VWR</td><td>JT9416-1</td><td></td></tr><tr><td>SU-DHL6</td><td>DSMZ</td><td>ACC 572</td><td></td></tr><tr><td>WSU-DLCL2</td><td>DSMZ</td><td>ACC 575</td><td></td></tr></tbody></table>

simultaneous imaging and flow-cytometry-based detection of multiple fluorescent senescence markers in therapy-induced senescent cancer cells

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, senescence is a fundamentally different machinery restraining&#160;propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated &#946;-galactosidase (SA-&#946;-gal), the DNA synthesis marker 5-ethynyl-2&#8242;-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (&#947;H2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.

A compensation matrix was generated using image analysis software by loading recorded data of single-color control samples. As shown in Supplemental Figure S1, a non-negligible (coefficient value ≥ 0.1) light spillover from EdU to C12FDG was detected with crosstalk coefficient value 0.248, while the crosstalk among other channels was not significant. Four different DLBCL cell lines were treated with 5 µg/mL MAF or 20 ng/mL DN to induce cellular senescence and analyzed using either conventional...

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

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

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Research

JoVE Journal

Cancer Research

This video illustrates a flow cytometry-based assay for quantifying therapy-induced senescent cancer cells with dual senescence markers. The detection and quantification of senescent cells involve using a β-galactosidase substrate. The substrate is cleaved by active lysosomal β-galactosidase within the senescent cells, producing a fluorescent product that, in conjunction with autofluorescent lipofuscin granules, marks the senescent cancer cells.

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

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A Sensitive Method to Quantify Senescent Cancer Cells

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Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated β-Galactosidase activity in single cells.

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

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Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

We describe a rapid staining method to perform multispectral imaging on frozen tissues.

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

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.

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

Multiplexed immunofluorescence enables sequential labeling of multiple antibody markers within a single tissue section to support spatial analysis of tissue architecture and cell populations. Here, a workflow for multiplex detection of senescence-associated, structural, and immune markers is presented, including manual and automated slide preparation, iterative imaging, and single-cell analysis using digital pathology software.

Multiplexed Immunofluorescence Assay for Spatial Assessment of Senescence Markers in vivo

An efficient screening protocol is presented for the identification of small molecules that promote astroglial differentiation in glioblastoma stem cells (GSCs). The assay is based on a stem cell differentiation reporter whereby the expression of the enhanced GFP (eGFP) is driven by the human GFAP promoter.

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

Learn imaging flow cytometry for detecting multiple senescence markers in cancer cells using DLBCL cell lines. This method enables rapid high-resolution analysis of fluorescent signals with spatial distribution data.

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells (Video) | JoVE Journal

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

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