Name: a quantitative measurement of reactive oxygen species and senescence-associated secretory phenotype in normal human fibroblasts during oncogene-induced senescence
Uploaded: 2018-08-12T13:00:00
Description: Watch this Scientific Journal Video about A Quantitative Measurement of Reactive Oxygen Species and Senescence-associated Secretory Phenotype in Normal Human Fibroblasts During Oncogene-induced Senesc

This work was supported by a grant from the National Research Foundation of Korea (2015R1D1A1A01060839) (to Young Yeon Kim) and by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2016R1A2B2008887, No. 2016R1A5A2007009) (to Jeanho Yun).

1. Inducing Oncogene-induced Senescence
<ol>
	<li>Preparing an H -RasV12 retrovirus
	<ol>
		<li>Coat the 100 mm culture dish by adding 2 mL of 0.001% poly-L-lysine/phosphate buffered saline (PBS) for 5 min at room temperature.</li>
		<li>Remove the poly-L-lysine solution using a glass pipette connected to a vacuum and wash the culture dish by adding 2 mL of 1x PBS.</li>
		<li>Plate 3 x 106 ecotropic BOSC-23 packaging cells on the coated culture dish with Dulbecco&#39;s Modified Eagle Medium ...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+serial+cultivation+of+human+diploid+cell+strains.">The serial cultivation of human diploid cell strains.</a> Experimental Cell Research. 25, 585-621 (1961).</li><li>Campisi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging,+cellular+senescence,+and+cancer.">Aging, cellular senescence, and cancer.</a> Annual Review of Physiology. 75, 685-705 (2013).</li><li>Braig, 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=Oncogene-induced+senescence+as+an+initial+barrier+in+lymphoma+development.">Oncogene-induced senescence as an initial barrier in lymphoma development.</a> Nature. 436 (7051), 660-665 (2005).</li><li>Michaloglou, C., 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=BRAFE600-associated+senescence-like+cell+cycle+arrest+of+human+naevi.">BRAFE600-associated senescence-like cell cycle arrest of human naevi.</a> Nature. 436 (7051), 720-724 (2005).</li><li>Collado, 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=Tumour+biology:+senescence+in+premalignant+tumours.">Tumour biology: senescence in premalignant tumours.</a> Nature. 436 (7051), 642 (2005).</li><li>Malaquin, N., Martinez, A., Rodier, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+the+senescence+secretome+under+control:+Molecular+reins+on+the+senescence-associated+secretory+phenotype.">Keeping the senescence secretome under control: Molecular reins on the senescence-associated secretory phenotype.</a> Experimental Gerontology. 82, 39-49 (2016).</li><li>Kuilman, T., Michaloglou, C., Mooi, W. 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C., 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=Ras+proteins+induce+senescence+by+altering+the+intracellular+levels+of+reactive+oxygen+species.">Ras proteins induce senescence by altering the intracellular levels of reactive oxygen species.</a> The Journal of Biological Chemistry. 274 (12), 7936-7940 (1999).</li><li>Chen, Q., Ames, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Senescence-like+growth+arrest+induced+by+hydrogen+peroxide+in+human+diploid+fibroblast+F65+cells.">Senescence-like growth arrest induced by hydrogen peroxide in human diploid fibroblast F65 cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (10), 4130-4134 (1994).</li><li>Dumont, P., 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=Induction+of+replicative+senescence+biomarkers+by+sublethal+oxidative+stresses+in+normal+human+fibroblast.">Induction of replicative senescence biomarkers by sublethal oxidative stresses in normal human fibroblast.</a> Free Radical Biology &amp; Medicine. 28 (3), 361-373 (2000).</li><li>Blander, G., de Oliveira, R. M., Conboy, C. M., Haigis, M., Guarente, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+dismutase+1+knock-down+induces+senescence+in+human+fibroblasts.">Superoxide dismutase 1 knock-down induces senescence in human fibroblasts.</a> The Journal of Biological Chemistry. 278 (40), 38966-38969 (2003).</li><li>Packer, L., Fuehr, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+oxygen+concentration+extends+the+lifespan+of+cultured+human+diploid+cells.">Low oxygen concentration extends the lifespan of cultured human diploid cells.</a> Nature. 267 (5610), 423-425 (1977).</li><li>Serra, V., von Zglinicki, T., Lorenz, M., Saretzki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+superoxide+dismutase+is+a+major+antioxidant+in+human+fibroblasts+and+slows+telomere+shortening.">Extracellular superoxide dismutase is a major antioxidant in human fibroblasts and slows telomere shortening.</a> The Journal of Biological Chemistry. 278 (9), 6824-6830 (2003).</li><li>Rodier, F., Campisi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four+faces+of+cellular+senescence.">Four faces of cellular senescence.</a> The Journal of Cell Biology. 192 (4), 547-556 (2011).</li><li>Munoz-Espin, D., Serrano, M. <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:+from+physiology+to+pathology.">Cellular senescence: from physiology to pathology.</a> Nature Reviews. Molecular Cell Biology. 15 (7), 482-496 (2014).</li><li>Tchkonia, T., Zhu, Y., van Deursen, J., Campisi, J., Kirkland, J. L. <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+the+senescent+secretory+phenotype:+therapeutic+opportunities.">Cellular senescence and the senescent secretory phenotype: therapeutic opportunities.</a> The Journal of Clinical Investigation. 123 (3), 966-972 (2013).</li><li>van Deursen, J. 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+role+of+senescent+cells+in+ageing.">The role of senescent cells in ageing.</a> Nature. 509 (7501), 439-446 (2014).</li><li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2(-Delta+Delta+C(T))+Method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.</a> Methods. 25 (4), 402-408 (2001).</li><li>Kim, Y. 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=Cooperation+between+p21+and+Akt+is+required+for+p53-dependent+cellular+senescence.">Cooperation between p21 and Akt is required for p53-dependent cellular senescence.</a> Aging Cell. 16 (5), 1094-1103 (2017).</li><li>Serrano, M., Lin, A. W., McCurrach, M. E., Beach, D., Lowe, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncogenic+ras+provokes+premature+cell+senescence+associated+with+accumulation+of+p53+and+p16INK4a.">Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a.</a> Cell. 88 (5), 593-602 (1997).</li><li>Wu, D., Yotnda, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+detection+of+reactive+oxygen+species+(ROS)+in+cancers.">Production and detection of reactive oxygen species (ROS) in cancers.</a> Journal of Visualized Experiments. (57), (2011).</li><li>Wojtala, 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=Methods+to+monitor+ROS+production+by+fluorescence+microscopy+and+fluorometry.">Methods to monitor ROS production by fluorescence microscopy and fluorometry.</a> Methods in Enzymology. 542, 243-262 (2014).</li><li>Duncan, F. 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=Age-associated+dysregulation+of+protein+metabolism+in+the+mammalian+oocyte.">Age-associated dysregulation of protein metabolism in the mammalian oocyte.</a> Aging Cell. 16 (6), 1381-1393 (2017).</li><li>Yang, L., Song, T., Chen, L., Soliman, H., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleolar+repression+facilitates+initiation+and+maintenance+of+senescence.">Nucleolar repression facilitates initiation and maintenance of senescence.</a> Cell Cycle. 14 (22), 3613-3623 (2015).</li><li>Coppe, J. P., 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=Senescence-associated+secretory+phenotypes+reveal+cell-nonautonomous+functions+of+oncogenic+RAS+and+the+p53+tumor+suppressor.">Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor.</a> PLoS Biology. 6 (12), 2853-2868 (2008).</li><li>Kosar, 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=Senescence-associated+heterochromatin+foci+are+dispensable+for+cellular+senescence,+occur+in+a+cell+type-+and+insult-dependent+manner+and+follow+expression+of+p16(ink4a).">Senescence-associated heterochromatin foci are dispensable for cellular senescence, occur in a cell type- and insult-dependent manner and follow expression of p16(ink4a).</a> Cell Cycle. 10 (3), 457-468 (2011).</li><li>Sharpless, N. E., Sherr, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forging+a+signature+of+in+vivo+senescence.">Forging a signature of in vivo senescence.</a> Nature Reviews. Cancer. 15 (7), 397-408 (2015).</li><li>Baker, D. J., 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=Opposing+roles+for+p16Ink4a+and+p19Arf+in+senescence+and+ageing+caused+by+BubR1+insufficiency.">Opposing roles for p16Ink4a and p19Arf in senescence and ageing caused by BubR1 insufficiency.</a> Nature Cell Biology. 10 (7), 825-836 (2008).</li><li>Baker, D. J., 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=Clearance+of+p16Ink4a-positive+senescent+cells+delays+ageing-associated+disorders.">Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders.</a> Nature. 479 (7372), 232-236 (2011).</li><li>Baar, M. P., 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=Targeted+Apoptosis+of+Senescent+Cells+Restores+Tissue+Homeostasis+in+Response+to+Chemotoxicity+and+Aging.">Targeted Apoptosis of Senescent Cells Restores Tissue Homeostasis in Response to Chemotoxicity and Aging.</a> Cell. 169 (1), 132-147 (2017).</li><li>Farr, J. N., 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=Targeting+cellular+senescence+prevents+age-related+bone+loss+in+mice.">Targeting cellular senescence prevents age-related bone loss in mice.</a> Nature Medicine. 23 (9), 1072-1079 (2017).</li><li>Chang, J., 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=Clearance+of+senescent+cells+by+ABT263+rejuvenates+aged+hematopoietic+stem+cells+in+mice.">Clearance of senescent cells by ABT263 rejuvenates aged hematopoietic stem cells in mice.</a> Nature Medicine. 22 (1), 78-83 (2016).</li><li>Yosef, R., 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=Directed+elimination+of+senescent+cells+by+inhibition+of+BCL-W+and+BCL-XL.">Directed elimination of senescent cells by inhibition of BCL-W and BCL-XL.</a> Nature Communications. 7, 11190 (2016).</li><li>Jeon, O. 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Here, we have presented methods for monitoring intracellular ROS levels during H-Ras-induced senescence in WI-38 normal human fibroblasts. Intracellular ROS levels in live cells can be measured quantitatively using the cell-permeable reagent DCF-DA and flow cytometry. Upon cellular uptake, DCF-DA is deacetylated by intracellular esterases and, subsequently, oxidized by ROS to form highly fluorescent 2&#39;,7&#39;-dichlorofluorescein (DCF). DCF fluorescence can be detected by flow cytometry using an FL1 detector (green fl...

More than 50 years ago, Hayflick and Moorhead revealed that normal cells enter irreversible growth arrest upon the exhaustion of their proliferative potential after a certain number of cell divisions1. This phenomenon is now known as replicative senescence and is believed to strongly correlate with organismal aging2. Although the progressive erosion of telomeres is considered a major cause of replicative senescence, various cellular stresses, such as DNA damage, oncogenic activation, and oxidative stress, have been reported to induce another type of cellular senescence called &#34;premature senescence&#34; or &#34;stress-induced senescence&#34;. Interestingly, premature senescence plays a potent tumor-suppressive role upon the activation of oncogenes such as H-Ras and BRAF. Studies of mouse models and human tissues have produced strong evidence that biomarkers of cell senescence were predominantly present in premalignant lesions where oncogenic Ras and BRAF are activated but were diminished in malignant cancers that developed from these lesions3,4,5. Beyond its role in aging and tumor suppression, cellular senescence has been shown in previous studies to play a role in various physiological processes, including wound healing, tissue repair, immune surveillance, and embryonic development6.
Although growth arrest has been extensively studied as a hallmark of cellular senescence7, a significant body of evidence suggests that intracellular reactive oxygen species (ROS) also contributes to cellular senescence8. The elevation of ROS levels during various types of cellular senescence, including replicative senescence and oncogene-induced senescence (OIS), was originally reported decades ago9,10. A more directly, exogenous treatment with a sublethal dose of H2O2 induces senescence11,12. The inhibition of ROS-scavenging enzymes, such as SOD1, also causes premature senescence13. In contrast, low ambient oxygen conditions and increasing ROS scavenging delay the onset of senescence10,14,15. These results undoubtedly indicate that ROS are important mediators or determinants of cellular senescence induction. However, how ROS contribute to the induction of cellular senescence and how ROS levels are elevated during cellular senescence require further investigation.
Recent studies have revealed that senescent cells have potent paracrine activities on neighboring cells and tissues through an SASP16,17. In aged tissue, senescent cells promote age-related tissue dysfunctions via many pathways through SASP in addition to an autonomous depletion of proliferative cells. Various proinflammatory factors, such as IL-6, IL-8, TGF&#946;, and matrix metalloproteinases (MMPs), secreted by senescent cells, cause age-related tissue dysfunctions through the impairment of tissue homeostasis, destruction of the tissue architecture, senescence of neighboring cells, and sterile inflammation18,19. However, SASPs can have beneficial effects depending on the biological context. In addition, the heterogenetic nature of SASPs depends on the senescent cell type and the cell stage, emphasizing the need for further research19.
Here, we describe rapid and sensitive cytometry-based techniques for assessing intracellular ROS levels during OIS. In addition, methods for the analysis of SASP factors using quantitative real-time polymerase chain reaction (qPCR) and ELISA are introduced.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>REAGENTS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>poly-L-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P2636</td>
			<td></td>
		</tr>
		<tr>
			<td>BOSC 23</td>
			<td>ATCC</td>
			<td>CRL-11269</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>GIBCO</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin/streptomycin</td>
			<td>wellgene</td>
			<td>LS202-02</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Hyclone</td>
			<td>SH30013.02</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>GIBCO</td>
			<td>12800-082</td>
			<td></td>
		</tr>
		<tr>
			<td>OPTI-MEM&#160;</td>
			<td>GIBCO</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>pBabe puro-H-RasV12&#160;</td>
			<td>Addgene</td>
			<td>1768</td>
			<td></td>
		</tr>
		<tr>
			<td>pGAG/pol</td>
			<td>Addgene</td>
			<td>14887</td>
			<td></td>
		</tr>
		<tr>
			<td>pVSVG</td>
			<td>Addgene</td>
			<td>1733</td>
			<td></td>
		</tr>
		<tr>
			<td>Turbofect</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0531</td>
			<td></td>
		</tr>
		<tr>
			<td>polybrene</td>
			<td>Sigma-Aldrich</td>
			<td>H9268</td>
			<td>8 mg/ml</td>
		</tr>
		<tr>
			<td>puromycin</td>
			<td>Sigma-Aldrich</td>
			<td>P8833</td>
			<td>2 mg/ml&#160;</td>
		</tr>
		<tr>
			<td>formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr>
			<td>5-bromo-4-chloro-3-indolyl &#946; D-galactopyranoside (X-gal)</td>
			<td>Sigma-Aldrich</td>
			<td>B4252</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium ferrocyanide</td>
			<td>Sigma-Aldrich</td>
			<td>B4252</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium ferricyanide</td>
			<td>Sigma-Aldrich</td>
			<td>P9387</td>
			<td></td>
		</tr>
		<tr>
			<td>trypsin-EDTA</td>
			<td>wellgene</td>
			<td>LS015-01</td>
			<td></td>
		</tr>
		<tr>
			<td>DCF-DA</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>D6883</td>
			<td>10 mM&#160;</td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Thermo Fisher Scientific</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>MMLV Reverse transcriptase</td>
			<td>Promega</td>
			<td>M1701</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Green PCR master 2X mix</td>
			<td>Takara</td>
			<td>PR820A</td>
			<td></td>
		</tr>
		<tr>
			<td>Random Primer</td>
			<td>Promega</td>
			<td>C118A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-pure distilled water</td>
			<td>Invitrogen</td>
			<td>10977015</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IL-6 ELISA assay</td>
			<td>PeproTech</td>
			<td>#900-TM16</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IL-8 ELISA assay</td>
			<td>PeproTech</td>
			<td>#900_TM18</td>
			<td></td>
		</tr>
		<tr>
			<td>EQUIPMENTS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 &#956;m syringe filter</td>
			<td>sartorius</td>
			<td>16555</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>BEMIS&#160;</td>
			<td>PM-996</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>NIKON</td>
			<td>TS100</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD Bioscience</td>
			<td>LSR Fortessa</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-4ml</td>
			<td>Merk Millipore</td>
			<td>UFC800324</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop spectrophotometer</td>
			<td>BioDrop</td>
			<td>80-3006-61</td>
			<td></td>
		</tr>
		<tr>
			<td>Real-time PCR System</td>
			<td>Applied Biosystems</td>
			<td>ABI Prism 7500</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA Reader</td>
			<td>Molecular Devices</td>
			<td>EMax microplate reader</td>
			<td></td>
		</tr>
	</tbody>
</table>

a quantitative measurement of reactive oxygen species and senescence-associated secretory phenotype in normal human fibroblasts during oncogene-induced senescence

Cellular senescence has been considered a state of irreversible growth arrest upon exhaustion of proliferative capacity or exposure to various stresses. Recent studies have extended the role of cellular senescence to various physiological processes, including development, wound healing, immune surveillance, and age-related tissue dysfunction. Although cell cycle arrest is a critical hallmark of cellular senescence, an increased intracellular reactive oxygen species (ROS) production has also been demonstrated to play an important role in the induction of cellular senescence. In addition, recent studies revealed that senescent cells exhibit potent paracrine activities on neighboring cells and tissues through a senescence-associated secretory phenotype (SASP). The sharp increase in interest regarding therapeutic strategies against cellular senescence emphasizes the need for a precise understanding of senescence mechanisms, including intracellular ROS and the SASP. Here, we describe protocols for quantitatively assessing intracellular ROS levels during H-Ras-induced cellular senescence using ROS-sensitive fluorescent dye and flow cytometry. In addition, we introduce sensitive techniques for the analysis of the induction of mRNA expression and secretion of SASP factors. These protocols can be applied to various cellular senescence models.

An example of H-Ras-induced senescence is shown in Figure 1. An infection of WI-38 normal human fibroblasts with the H-RasV12 retrovirus induced dramatic morphological changes (Figure 1B). In addition, as shown in Figure 1C, SA &#946;-gal staining activity was remarkably increased upon H-RasV12 expression. More than 70% of the cells showed SA &#946;-gal staining activity 6 d after the H-RasV12 retrov...

Watch this Scientific Journal Video about A Quantitative Measurement of Reactive Oxygen Species and Senescence-associated Secretory Phenotype in Normal Human Fibroblasts During Oncogene-induced Senesc

A Quantitative Measurement of Reactive Oxygen Species and Senescence-associated Secretory Phenotype in Normal Human Fibroblasts During Oncogene-induced Senescence

Intracellular ROS has been shown to play an important role in the induction of cellular senescence. Here, we describe a sensitive assay for quantifying ROS levels during cellular senescence. We also provide protocols for assessing the senescence-associated secretory phenotype, which reportedly contributes to various age-related dysfunctions.

Cellular senescence has been considered a state of irreversible growth arrest upon exhaustion of proliferative capacity or exposure to various stresses. ...

The overall goal of this procedure is to provide sensitive techniques for monitoring intracellular ROS levels and senescence-associated secretory phenotype, or SASP, during cellular senescence. Here, we demonstrate that these methods successfully reveal the increases in the levels of intracellular ROS and the SASP factors IL-6 and IL-8 during H-Ras-induced senescence in WI-38 normal human fibroblasts. These techniques allow monitoring changes in intracellular ROS levels during cellular senescence and the analysis of the expression of SASP factors.The main advantage of this method is that they can be applied to many different cellular senescence models. Therefore, this method can ultimately help us to understand the role of ROS and SASP in cellular senescence and to study the underlying regulatory mechanisms. Demonstrating the procedure will be Young Yeon Kim and Jee-Hyun Um from my laboratory.Begin this procedure by preparing the H-Ras retrovirus. First, seed ecotropic BOSC cells in a poly-L-lysine-coated culture dish, and culture at 37 degrees Celsius in a tissue culture incubator for one day. On the next day, transfect the BOSC cells with H-Ras retroviral DNA and packaging DNAs using transfection reagent according to the manufacturer's instructions.After eight hours of transfection, remove the transfection media and add eight milliliters of fresh media. Collect the media containing viral particles 48 hours later, and remove cellular debris by centrifugation. Next, filter the virus-containing media with a 0.45-micrometer syringe filter.Plate WI-38 cells in a 60-millimeter culture dish one day before harvest of H-Ras retrovirus, and culture for one day at 37 degrees Celsius in an incubator. On the next day, remove the growth medium, and add one milliliter of H-Ras virus media. Add an additional one milliliter of growth medium containing two microliters of polybrene stock solution, and incubate for one day at CO2 incubator.At 24 hours after infection, remove the virus media, wash the cells twice with PBS, and add the growth media. To remove non-infected cells, treat the cells for two days with two micrograms per milliliter of puromycin. Prepare fresh SA beta-gal staining solution by diluting the stock solution.Remove the culture media, and wash the cells once with PBS. Add fixation solution containing formaldehyde, and incubate for five minutes. Remove the fixation solution from the cells, and wash with PBS.Add freshly prepared SA beta-gal staining solution to cover the dish. Seal the dish with Parafilm to avoid drying the sample, and incubate at 37 degrees Celsius in an incubator without CO2. Check the staining status of the cells under microscope on the next day.SA beta-gal-positive cells will appear blue in the perinuclear region. Acquire an image of SA beta-gal staining with a CCD camera, and calculate the percentage of SA beta-gal-stained cells by counting the number of SA beta-gal-positive cells among at least 200 total cells. Plate two times 10 to the fifth WI-38 cells on a 60-millimeter culture dish, and induce senescence by infecting the cells with H-Ras retrovirus as described previously.At the desired time point for measurement of ROS, remove the growth medium and wash once with PBS. Detach the cells by treatment with trypsin/EDTA solution, and determine the cell count. Transfer one times 10 to the fifth cells into a 15-milliliter conical tube, and collect the cells by centrifugation.Remove the growth medium carefully, and add one milliliter of freshly prepared DCF-DA staining medium. Carefully resuspend the cell pellet, and incubate at 37 degrees Celsius for 30 minutes in the dark. Collect the cells by centrifugation and wash once with PBS.Resuspend the cells with one milliliter of PBS, and transfer the samples into a FACS tube. The DCF-DA fluorescence signal can be measured using a flow cytometer equipped with a 488-nanometer blue laser. First, run a non-stained control cell sample.In the dot plot of the forward scatter versus side scatter, select live cells and eliminate dead cells and cell debris by using a gating tool. Next, in the histogram displaying DCF-DA fluorescence signal, adjust the blue laser voltage to place the peak from the control cells at the left edge. Now, run each sample stained with DCF-DA, and record at least 10, 000 events.Induce senescence by infecting the cells with H-Ras retrovirus as described previously. At one day before harvest, wash the cells twice with PBS. Add three milliliters of DMEM without FBS, and culture the cells at 37 degrees Celsius in a tissue culture incubator for one day.After 24 hours, collect the conditioned medium, and store at negative 80 degrees Celsius for subsequent ELISA. Trypsinize the cells, and harvest the cells in a 1.5-milliliter microcentrifuge tube by centrifugation. Prepare total RNA using the conventional RNA extraction method, and generate cDNA from two micrograms of total RNA through a reverse transcription reaction.For real-time PCR, prepare SYBR Green PCR Master Mix for all reactions containing the forward and reverse primers for IL-6 or IL-8. Prepare the reaction mixture for each sample containing actin primers as an internal control. Add 48 microliters of Master Mix and two microliters of cDNA products diluted one to three to each well in a 96-well optical qPCR plate.Perform each reaction in triplicates. Set up the real-time PCR reaction program on the thermal cycler, and perform real-time PCR. Upon completion of the run, collect the threshold cycle values, or CT values, and calculate the mRNA levels of IL-6 or IL-8 after normalization to the actin levels.Thaw conditioned media harvested previously from senescent WI-38 cells. Remove cell debris by centrifugation at 500 g for five minutes. Concentrate the conditioned medium by 10-fold using a centrifugal filter with a pore size of 3, 000 dalton.Perform the ELISA with the appropriate IL-6 or IL-8 ELISA kit. First, coat the ELISA plate well with diluted IL-6 or IL-8 antibody by incubating the plate overnight at room temperature. After washing each well four times, add blocking buffer to each well, and incubate for one hour.Wash the plate four times. Add either serially diluted ELISA standards or concentrated conditioned medium samples, and incubate for two hours. After washing the plate, incubate the samples with a secondary antibody and streptavidin-HRP conjugate sequentially.Add 100 microliters of TMB substrate solution to each well, and incubate at room temperature for 20 minutes to allow for color development. Stop the reaction by adding stop solution. Read the absorbance of each well with an ELISA reader at 450 nanometer.Plot the standard curve for the generated standards. Calculate the concentrations of IL-6 and IL-8 in the conditioned medium according to the standard curves. Plot the results after normalization to the cell count.Infection of WI-38 normal human fibroblasts with H-Ras retrovirus induced dramatic changes in cellular morphology. SA beta-gal staining activity was observed in more than 70%of the cells, indicating that H-Ras expression induces cellular senescence in WI-38 cells within six days. DCF-DA staining analysis reveals that H-Ras expression also induces a significant increase in intracellular ROS levels.The increase in intracellular ROS levels is observed as early as two days after H-Ras expression and is maintained until the six-day time point. Real-time PCR analysis of the representative senescence-associated secretory factors IL-6 and IL-8 reveals that mRNA expression of IL-6 and IL-8 is remarkably increased in senescent WI-38 cells. ELISAs of conditioned medium confirmed that IL-6 and IL-8 secretion is also increased in H-Ras-induced senescent cells.Inducing cellular senescence through oncogene expression, DNA damage, or oxidative stress usually takes six to eight days. Once the cells are ready, ROS measurement can be conducted within two hour if performed properly. After mastery of these techniques, analysis of the mRNA and protein levels of SASP factors can be completed within one day.When analyzing ROS levels and SASP induction, it is important to confirm cellular senescence through SA beta-gal staining and additional method if necessary. In addition, it is critical to remember that the increase of ROS occurs in the early phase of senescence, whereas the development of SASP is detectable only after senescence is fully established. These techniques can be applied to a wide variety of cellular senescence models.After watching this video, you should have a good understanding of how to analyze ROS levels and SASP induction during cellular senescence.

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