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

Detta arbete var stöds av ett bidrag från den National Research Foundation of Korea (2015R1D1A1A01060839) (till ung Yeon Kim) och National Research Foundation i Korea (NRF) bidrag finansieras av Korea regeringen (MSIT) (nr 2016R1A2B2008887, Nr. 2016R1A5A2007009) (till Jeanho Yun).

1. inducerande onkogen-inducerad åldras
<ol><li>
Förbereda en H-RasV12 retrovirus
	<ol>
<li>Coat 100 mm kultur skålen genom att lägga till 2 mL av 0,001% poly-L-lysin/fosfat buffrad saltlösning (PBS) för 5 min i rumstemperatur.</li>
		<li>Ta bort den poly-L-lysin lösningen med glas pipett ansluten till ett vakuum och tvätta kultur skålen genom att tillsätta 2 mL 1 x PBS.</li>
		<li>Plate 3 x 106 ecotropic BOSC-23 förpackningar celler på bestruket kulturen skålen med Dulbeccos mo...

<ol>
	<li>Hayflick, L., Moorhead, P. 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. J., Peeper, D. 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+essence+of+senescence.">The essence of senescence.</a> Genes &amp; Development. 24 (22), 2463-2479 (2010).</li><li>Lu, T., Finkel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radicals+and+senescence.">Free radicals and senescence.</a> Experimental Cell Research. 314 (9), 1918-1922 (2008).</li><li>Furumoto, K., Inoue, E., Nagao, N., Hiyama, E., Miwa, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+telomere+shortening+is+slowed+down+by+enrichment+of+intracellular+vitamin+C+via+suppression+of+oxidative+stress.">Age-dependent telomere shortening is slowed down by enrichment of intracellular vitamin C via suppression of oxidative stress.</a> Life Sciences. 63 (11), 935-948 (1998).</li><li>Lee, A. 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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Här har vi presenterat metoder för att övervaka intracellulära ROS nivåer under H-Ras-inducerad åldras i WI-38 normala mänskliga fibroblaster. Intracellulära ROS nivåer i levande celler kan mätas kvantitativt med hjälp av cell-permeable reagensen DCF-DA och flödescytometri. På cellernas upptag, DCF-DA deacetylated av intracellulära esteraser och därefter oxideras av ROS bildar starkt fluorescerande 2', 7'-dichlorofluorescein (DCF). DCF fluorescens kan upptäckas av flödescytometri med en FL1 detektor (gr?...

Mer än 50 år sedan visade att normala celler in irreversibel tillväxt gripandet vid konsumtion av deras proliferativ potential efter ett visst antal celldelningar1Hayflick och Moorhead. Detta fenomen kallas nu replikationsförmåga åldras och tros starkt korrelerar med organismers åldrande2. Även om en gradvis urholkning av telomerer anses vara en viktig orsak till replikationsförmåga åldras, har olika cellulära påfrestningar, som DNA skador, onkogena aktivering och oxidativ stress, rapporterats inducera en annan typ av cellulära åldras kallas ”förtida åldrande” eller ”stressinducerade åldras”. Intressant, spelar för tidigt åldrande en potent tumör-suppressiv roll vid aktivering av onkogener som H-Ras och BRAF. Studier av musmodeller och mänskliga vävnader har producerat starka bevis som biomarkörer för cellen åldras fanns huvudsakligen i premaligna lesioner där onkogena Ras och BRAF aktiveras men minskades i elakartade cancerformer som utvecklats från dessa lesioner3,4,5. Utöver sin roll i åldrande och tumör dämpning, har cellulära åldras visats i tidigare studier att spela en roll i olika fysiologiska processer, inklusive sårläkning, vävnad reparera, immun övervakning och embryonal utveckling6.
Även om tillväxt gripandet har studerats som ett kännetecken för cellulära åldras7, tyder en betydande mängd bevis det intracellulära reaktiva syreradikaler (ROS) bidrar också till cellulära åldras8. Höjden av ROS nivåer under olika typer av cellulära åldras, inklusive replikationsförmåga åldras och onkogen-inducerad åldras (OIS), rapporterades ursprungligen decennier sedan9,10. En mer direkt, exogena behandling med en subletala dos av H2O2 inducerar åldras11,12. Hämning av ROS-rensning enzymer, såsom SOD1, orsakar också tidig åldras13. Däremot låg omgivande Syreförhållandena och öka ROS rensning försening uppkomsten av åldras10,14,15. Resultaten visar utan tvekan att ROS är viktiga medlare eller bestämningsfaktorerna för cellulära åldras induktion. Men hur ROS bidrar till induktion av cellulära åldras och hur ROS-nivåerna är förhöjda under cellulärt åldrande kräver ytterligare utredning.
Senare studier visat att senescent celler har potent parakrin aktiviteter på angränsande celler och vävnader genom en SASP16,17. I år vävnad främja senescent celler åldersrelaterade vävnad dysfunktioner via många vägar genom SASP förutom en autonom uttömning av proliferativ celler. Olika proinflammatoriska faktorer, till exempel IL-6, IL-8, TGFβ och matrix metalloproteinaser (MMP), utsöndras av senescent celler, orsaka åldersrelaterade vävnad dysfunktioner genom nedskrivning av vävnad homeostas, förstörelse av vävnad arkitektur, åldras av närliggande celler och steril inflammation18,19. Dock kan SASPs ha gynnsamma effekter beroende på de biologiska sammanhanget. Dessutom beror heterogenetic arten av SASPs på den senescent celltypen och cellen scenen, betonar behovet av ytterligare forskning19.
Här beskriver vi snabba och känsliga flödescytometri-baserad teknik för att bedöma intracellulära ROS nivåer under OIS. Dessutom introduceras metoder för analys av SASP faktorer med hjälp av kvantitativ realtids polymeras-kedjereaktion (qPCR) och ELISA.

<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

Cellulära åldras har ansetts vara ett tillstånd av irreversibel tillväxt gripandet vid konsumtion av proliferativ kapacitet eller exponering för olika påfrestningar. Nyligen genomförda studier har utökat rollen av cellulära åldras olika fysiologiska processer, inklusive utveckling, sårläkning, immun övervakning och åldersrelaterade vävnad dysfunktion. Även om cellcykelarrest är ett avgörande kännetecken för cellulära åldras, har en ökad intracellulär reaktivt syre arter (ROS) produktion också visats spela en viktig roll i induktion av cellulära åldras. Senare studier visade dessutom att senescent celler uppvisar potent parakrin aktiviteter på angränsande celler och vävnader genom en åldras-associerade sekretoriska fenotyp (SASP). Den kraftiga ökningen intresse angående terapeutiska strategier mot cellulära åldras betonar behovet av en exakt förståelse åldras mekanismer, inklusive intracellulära ROS och SASP. Här beskriver vi protokoll för att kvantitativt bedöma intracellulära ROS nivåer under H-Ras-inducerad cellulära åldras med ROS-känsliga fluorescerande färgämne och flödescytometri. Dessutom introducerar vi känsliga tekniker för analys av induktion av mRNA uttryck och utsöndring av SASP faktorer. Dessa protokoll kan tillämpas på olika cellulära åldras modeller.

Ett exempel på H-Ras-inducerad åldras visas i figur 1. En infektion i WI-38 normala mänskliga fibroblaster med den H-RasV12 retrovirus inducerad dramatiska morfologiska förändringar (figur 1B). Dessutom, som visas i figur 1 c, var SA β-gal färgning aktivitet anmärkningsvärt ökade på H-RasV12 uttryck. Mer än 70% av cellerna visade SA β-gal färgning aktivitet 6 d efter H-RasV12 retrovirus...

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

Intracellulära ROS har visat sig spela en viktig roll i induktion av cellulära åldras. Här beskriver vi en känslig analys för att kvantifiera ROS nivåer under cellulära åldras. Vi tillhandahåller även protokoll för att bedöma den åldras-associerade sekretoriska fenotyp, som enligt uppgift bidrar till olika åldersrelaterade dysfunktioner.

En kvantitativ mätning av reaktiva syreradikaler och åldras-associerade sekretoriska fenotyp i normala mänskliga fibroblaster under onkogen-inducerad åldras

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

Research

JoVE Journal

Developmental Biology

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

Isolering och Kvantitativ Immuncytokemiska karakterisering av primära myogena celler och fibroblaster från Human skelettmuskulatur

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

Etablering av proliferativ tetraploid celler från icke-transforme humanfibroblaster

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to ...

Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa

Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa

Utvärdering av intracellulära läge av reaktiva syreradikaler i Solea Senegalensis spermier

Oxidative stress is one of the important factors in decreasing sperm quality. Developing efficient protocols for detecting reactive oxygen species (ROS) ...

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Ljusmedierad reversibel modulering av den mitogenaktiverade proteinkinasvägen under celldifferentiering och<em&gt; Xenopus</em&gt; Embryonisk utveckling

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early ...

A Method for Characterizing Embryogenesis in Arabidopsis

A Method for Characterizing Embryogenesis in Arabidopsis

En metod för att karaktärisera embryogenes i Arabidopsis

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Utvärdering av nervskade-inducerad åldras och In Vivo omprogrammering i skelettmuskulaturen

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Cell Aggregation analyser att utvärdera bindningen av Drosophila skåran med Trans-ligander och dess hämning av Cis-ligander

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Realtid mätning av epiteliala barriären permeabilitet i Human Intestinal Organoids

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

Induction and Validation of Cellular Senescence in Primary Human Cells

Induktion och validering av cellulära åldras i primära mänskliga celler

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

RNA-baserade omprogrammering av mänskliga primära fibroblaster till inducerade pluripotenta stamceller

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...