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

Dette arbejde blev støttet af tilskud fra National Research Foundation Korea (2015R1D1A1A01060839) (til unge Yeon Kim) og National Research Foundation af Korea (NRF) tilskud finansieret af Korea regering (MSIT) (Nej 2016R1A2B2008887, nr. 2016R1A5A2007009) (til Jeanho Yun).

1. inducerende onkogen-induceret gulne
<ol><li>
Forberede en H-RasV12 retrovirus
	<ol>
<li>Frakke 100 mm kultur parabol ved tilsætning 2 mL af 0,001% poly-L-lysin/phosphat bufferet saltvand (PBS) i 5 min ved stuetemperatur.</li>
		<li>Fjerne den poly-L-lysin løsning ved hjælp af et glas pipette tilsluttet en vacuum og vaske kultur parabol ved tilsætning 2 mL 1 x PBS.</li>
		<li>Plade 3 x 106 ecotropic BOSC-23 emballage celler på den coatede kultur fad med Dulbeccos modificerede Eagle Me...

<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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Her har vi præsenteret metoder for overvågning intracellulære ROS niveauer under H-Ras-induceret gulne i WI-38 normale humane fibroblaster. Intracellulære ROS niveauer i levende celler kan måles kvantitativt ved hjælp af celle-gennemtrængelige reagenset DCF-DA og flow flowcytometri. På cellulære optagelse, er DCF-DA deacetylated af intracellulære esteraser og efterfølgende oxideres af indtægtsordrer, der skal danne stærkt fluorescerende 2', 7'-dichlorofluorescein (DCF). DCF fluorescens kan påvises ved flowc...

Mere end 50 år siden, afslørede Hayflick og Moorhead at normale celler Angiv irreversible vækst anholdelse efter konsumption af deres proliferativ potentiale efter et bestemt antal celledelinger1. Dette fænomen kaldes nu replicative gulne og menes at kraftigt korrelerer med kropsligt aging2. Selv om en gradvis udhuling af telomerer betragtes som en væsentlig årsag til replicative gulne, er forskellige cellulære understreger, såsom DNA skader, muterede aktivering og oxidativ stress, blevet rapporteret til at fremkalde en anden type af cellulære gulne kaldet "tidlig ældning" eller "stress-induceret gulne". Det er interessant, spiller for tidlig ældning en potent tumor-undertrykkende rolle ved aktivering af onkogener som H-Ras og BRAF. Undersøgelser af musemodeller og humant væv have produceret stærke beviser, som biomarkører for celle ældning fandtes overvejende i præmaligne læsioner hvor muterede Ras og BRAF er aktiveret, men var faldet i ondartede kræftformer, der er udviklet fra Disse læsioner3,4,5. Ud over sin rolle i aging og tumor undertrykkelse, har cellulære gulne vist i tidligere undersøgelser til at spille en rolle i forskellige fysiologiske processer, herunder sårheling, væv reparation, immun overvågning og fosterudviklingen6.
Selv om væksten anholdelsen er blevet grundigt undersøgt som kendetegnende for cellulære gulne7, tyder en betydelig række indicier på, at intracellulære reaktive ilt arter (ROS) bidrager også til cellulære gulne8. Udvidelse af ROS niveauer under forskellige typer af cellulære gulne, herunder replicative gulne og onkogen-induceret gulne (OIS), blev oprindeligt rapporteret årtier siden9,10. En mere direkte, eksogene behandling med en subletale dosis af H2O2 inducerer gulne11,12. Hæmning af ROS-scavenging enzymer, såsom SOD1, også forårsager for tidlig ældning13. Derimod lav omgivende ilt betingelser og stigende ROS skylleluften forsinkelse udbrud af gulne10,14,15. Disse resultater viser utvivlsomt, at ROS er vigtigt mæglere eller determinanter for cellulære gulne induktion. Men hvordan ROS bidrage til induktion af cellulære gulne og hvordan ROS niveauer er forhøjede under cellulære gulne kræve yderligere undersøgelse.
Nylige undersøgelser har afsløret, at senescent celler har potent paracrine aktiviteter på tilstødende celler og væv gennem en SASP16,17. I alderen væv fremme senescent celler aldersrelaterede væv dysfunktioner via mange stier gennem SASP ud over en selvstyrende udtynding af proliferativ celler. Forskellige proinflammatoriske faktorer, såsom IL-6, IL-8, TGFβ og matrix metalloproteinases (MMPs), udskilles af senescent celler, forårsage aldersrelaterede væv dysfunktioner gennem forringelse af væv homøostase, ødelæggelse af væv arkitektur, ældning af tilstødende celler og steril betændelse18,19. SASPs kan dog have gavnlige virkninger afhængigt af de biologiske kontekst. Heterogenetic karakter af SASPs afhænger Derudover senescent celletype og celle stadium, understreger behovet for yderligere forskning19.
Her beskriver vi hurtige og følsomme flowcytometri-baserede teknikker til vurdering af intracellulære ROS niveauer under OIS. Derudover introduceres metoder til analyse af SASP faktorer ved hjælp af kvantitative real-time polymerase kædereaktion (qPCR) og 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

Cellulære gulne har været betragtet som en tilstand af irreversible vækst anholdelse efter konsumption af proliferativ kapacitet eller udsættes for forskellige belastninger. Nylige undersøgelser har udvidet rolle i cellulære gulne til forskellige fysiologiske processer, herunder udvikling, sårheling, immun overvågning og aldersrelaterede væv dysfunktion. Selv om celle cyklus anholdelse er en kritisk kendetegnende for cellulære gulne, har en øget intracellulær reaktive ilt arter (ROS) produktion også påvist for at spille en vigtig rolle i induktion af cellulære ældning. Nylige undersøgelser afslørede desuden, at senescent celler udviser potent paracrine aktiviteter på tilstødende celler og væv gennem en gulne-associerede sekretoriske fænotype (SASP). Den kraftige stigning i interesse vedrørende terapeutiske strategier mod cellulære gulne understreger behovet for en præcis forståelse af gulne mekanismer, herunder intracellulære ROS og SASP. Her beskriver vi protokoller til kvantitativt at vurdere intracellulære ROS niveauer under H-Ras-induceret cellulære ældning ved hjælp af ROS-følsomme fluorescerende farvestof og flowcytometri. Derudover introducerer vi følsomme teknikker til analyse af induktion af mRNA udtryk og sekretion af SASP faktorer. Disse protokoller kan anvendes til forskellige cellulære gulne modeller.

Et eksempel på H-Ras-induceret gulne er vist i figur 1. En infektion i WI-38 normale humane fibroblaster med H-RasV12 retrovirus induceret dramatiske morfologiske ændringer (figur 1B). Som vist i figur 1 c, øgedes SA β-gal farvning aktivitet desuden bemærkelsesværdigt ved H-RasV12 udtryk. Mere end 70% af celler viste SA β-gal farvning aktivitet 6 d efter H-RasV12 retrovirus infektion, der angi...

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

Intracellulære ROS har vist sig at spille en vigtig rolle i induktion af cellulære ældning. Her, beskriver vi en følsom analyse for kvantificering ROS niveauer under cellulære ældning. Vi leverer også protokoller for at vurdere den gulne-associerede sekretoriske fænotype, som efter sigende bidrager til forskellige aldersrelaterede dysfunktioner.

En kvantitativ måling af reaktive ilt arter og gulne-associerede sekretoriske fænotype i normale humane fibroblaster under onkogen-induceret gulne

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 og kvantitativ Immuncytokemisk karakterisering af primære myogene celler og fibroblaster fra human skeletmuskel

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 af proliferativ Tetraploide Celler fra transformerede humane fibroblaster

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

Evaluering af intracellulær placering af reaktive ilt arter i Solea Senegalensis sædcellerne

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

Lysmedieret reversibel modulering af den mitogenaktiverede proteinkinasevej under celledifferentiering og<em&gt; Xenopus</em&gt; Embryonudvikling

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 metode til kendetegner embryogenese 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

Evaluering af skade-induceret gulne og In Vivo omprogrammering i den skeletmuskulatur

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

Celle sammenlægning Assays til at evaluere bindingen af Drosophila Notch med Trans-ligander og dens hæmning af 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

Real-time måling af epitel barriere permeabilitet i menneskelige tarm 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 og validering af cellulære gulne i primære humane 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-baserede omprogrammering af menneskelige primære fibroblaster til induceret pluripotente 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 ...