Name: assessing lysosomal alkalinization in the intestine of live caenorhabditis elegans
Uploaded: 2018-04-13T14:30:00
Description: Watch this Scientific Journal Video about Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans at JoVE.com

Vi vil gjerne takke Caenorhabditis genetikk Center for stammer, naturfag og Engineering Research Council (NSERC), og Canada grunnlaget for innovasjon (CFI) for finansiering. Vi vil gjerne takke Dr. Lizhen Chen (avdeling av cellen systemer og anatomi, UT helse San Antonio) for å tillate ubegrenset bruk av hennes lab fasiliteter for alle C. elegans eksperimenter som Dr. Exing Wang (knytte direktør, optisk Imaging anlegg UT helse San Antonio) for hjelp med AC confocal mikroskopi. Vi vil også gjerne takke Dr. Myron Ignatius for å gi støtte og oppmuntring til rette video skyte.

1. flekken og Image intestinal lysosomer
<ol><li>Frø Rundormer vekst medier (NGM) plater med OP50
	<ol>
<li>Forberede NGM plater som anbefalte protokollen19 og la lukket platene tørke 2 dager ved romtemperatur.</li>
		<li>Vaksinere OP50 bakterier i sterilt Luria kjøttkraft (LB) buljong og vokse i et risting inkubator eller vann bad på 37 ° C i 36 h eller til OD er mellom 0,2 og 0,4. Unngå å bruke en bakteriell kultur med OD > 1 eller OD < 0,2.</li>
		<li>Når NGM platene ...

<ol>
	<li>Erjavec, N., Larsson, L., Grantham, J., Nystrom, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+aging+and+failure+to+segregate+damaged+proteins+in+Sir2+mutants.">Accelerated aging and failure to segregate damaged proteins in Sir2 mutants.</a> Genes Dev. 21 (19), 2410-2421 (2007).</li><li>Madeo, F., Eisenberg, T., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+for+the+avoidance+of+neurodegeneration.">Autophagy for the avoidance of neurodegeneration.</a> Genes Dev. 23 (19), 2253-2259 (2009).</li><li>Rubinsztein, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+intracellular+protein-degradation+pathways+in+neurodegeneration.">The roles of intracellular protein-degradation pathways in neurodegeneration.</a> Nature. 443 (7113), 780-786 (2006).</li><li>Cohen, E., Bieschke, J., Perciavalle, R. M., Kelly, J. W., Dillin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposing+activities+protect+against+age-onset+proteotoxicity.">Opposing activities protect against age-onset proteotoxicity.</a> Science. 313 (5793), 1604-1610 (2006).</li><li>Cuervo, A. 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=Autophagy+and+aging:+the+importance+of+maintaining+&amp;#34;clean&amp;#34;+cells.">Autophagy and aging: the importance of maintaining &amp;#34;clean&amp;#34; cells.</a> Autophagy. 1 (3), 131-140 (2005).</li><li>Harrington, A. J., Knight, A. L., Caldwell, G. A., Caldwell, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+as+a+model+system+for+identifying+effectors+of+alpha-synuclein+misfolding+and+dopaminergic+cell+death+associated+with+Parkinson's+disease.">Caenorhabditis elegans as a model system for identifying effectors of alpha-synuclein misfolding and dopaminergic cell death associated with Parkinson's disease.</a> Methods. 53 (3), 220-225 (2011).</li><li>Muchowski, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+misfolding,+amyloid+formation,+and+neurodegeneration:+a+critical+role+for+molecular+chaperones?.">Protein misfolding, amyloid formation, and neurodegeneration: a critical role for molecular chaperones?.</a> Neuron. 35 (1), 9-12 (2002).</li><li>Cuervo, A. M., Dice, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+decline+in+chaperone-mediated+autophagy.">Age-related decline in chaperone-mediated autophagy.</a> J Biol Chem. 275 (40), 31505-31513 (2000).</li><li>Tonoki, 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=Genetic+evidence+linking+age-dependent+attenuation+of+the+26S+proteasome+with+the+aging+process.">Genetic evidence linking age-dependent attenuation of the 26S proteasome with the aging process.</a> Mol Cell Biol. 29 (4), 1095-1106 (2009).</li><li>Squier, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+and+protein+aggregation+during+biological+aging.">Oxidative stress and protein aggregation during biological aging.</a> Exp Gerontol. 36 (9), 1539-1550 (2001).</li><li>Sarkar, S., 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=Small+molecules+enhance+autophagy+and+reduce+toxicity+in+Huntington's+disease+models.">Small molecules enhance autophagy and reduce toxicity in Huntington's disease models.</a> Nat Chem Biol. 3 (6), 331-338 (2007).</li><li>Glick, D., Barth, S., Macleod, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy:+cellular+and+molecular+mechanisms.">Autophagy: cellular and molecular mechanisms.</a> J Pathol. 221 (1), 3-12 (2010).</li><li>Boya, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysosomal+function+and+dysfunction:+mechanism+and+disease.">Lysosomal function and dysfunction: mechanism and disease.</a> Antioxid Redox Signal. 17 (5), 766-774 (2012).</li><li>Kim, D. K., 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=Anti-aging+treatments+slow+propagation+of+synucleinopathy+by+restoring+lysosomal+function.">Anti-aging treatments slow propagation of synucleinopathy by restoring lysosomal function.</a> Autophagy. 12 (10), 1849-1863 (2016).</li><li>Vila, M., Bove, J., Dehay, B., Rodriguez-Muela, N., Boya, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysosomal+membrane+permeabilization+in+Parkinson+disease.">Lysosomal membrane permeabilization in Parkinson disease.</a> Autophagy. 7 (1), 98-100 (2011).</li><li>Cuervo, A. M., Dice, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+do+intracellular+proteolytic+systems+change+with+age?.">How do intracellular proteolytic systems change with age?.</a> Front Biosci. 3, d25-d43 (1998).</li><li>Cuervo, A. M., Dice, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=When+lysosomes+get+old.">When lysosomes get old.</a> Exp Gerontol. 35 (2), 119-131 (2000).</li><li>Gachet, Y., Codlin, S., Hyams, J. S., Mole, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=btn1,+the+Schizosaccharomyces+pombe+homologue+of+the+human+Batten+disease+gene+CLN3,+regulates+vacuole+homeostasis.">btn1, the Schizosaccharomyces pombe homologue of the human Batten disease gene CLN3, regulates vacuole homeostasis.</a> J Cell Sci. 118 (Pt 23), 5525-5536 (2005).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook. , 1-11 (2006).</li><li>Preston, R. A., Murphy, R. F., Jones, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+vacuolar+pH+in+yeast+and+identification+of+acidification-defective+mutants.">Assay of vacuolar pH in yeast and identification of acidification-defective mutants.</a> Proc Natl Acad Sci U S A. 86 (18), 7027-7031 (1989).</li><li>Pringle, J. 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=Fluorescence+microscopy+methods+for+yeast.">Fluorescence microscopy methods for yeast.</a> Methods Cell Biol. 31, 357-435 (1989).</li><li>Baxi, K., Ghavidel, A., Waddell, B., Harkness, T. A., de Carvalho, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Lysosomal+Function+by+the+DAF-16+Forkhead+Transcription+Factor+Couples+Reproduction+to+Aging+in+Caenorhabditis+elegans.">Regulation of Lysosomal Function by the DAF-16 Forkhead Transcription Factor Couples Reproduction to Aging in Caenorhabditis elegans.</a> Genetics. 207 (1), 83-101 (2017).</li><li>Colacurcio, D. J., Nixon, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disorders+of+lysosomal+acidification-The+emerging+role+of+v-ATPase+in+aging+and+neurodegenerative+disease.">Disorders of lysosomal acidification-The emerging role of v-ATPase in aging and neurodegenerative disease.</a> Ageing Res Rev. 32, 75-88 (2016).</li><li>Kang, H. T., 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=Chemical+screening+identifies+ATM+as+a+target+for+alleviating+senescence.">Chemical screening identifies ATM as a target for alleviating senescence.</a> Nat Chem Biol. 13 (6), 616-623 (2017).</li><li>Arrasate, M., Mitra, S., Schweitzer, E. S., Segal, M. R., Finkbeiner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inclusion+body+formation+reduces+levels+of+mutant+huntingtin+and+the+risk+of+neuronal+death.">Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death.</a> Nature. 431 (7010), 805-810 (2004).</li><li>Brunk, U. T., Terman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial-lysosomal+axis+theory+of+aging:+accumulation+of+damaged+mitochondria+as+a+result+of+imperfect+autophagocytosis.">The mitochondrial-lysosomal axis theory of aging: accumulation of damaged mitochondria as a result of imperfect autophagocytosis.</a> Eur J Biochem. 269 (8), 1996-2002 (2002).</li><li>Gerland, L. 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=Autolysosomes+accumulate+during+in+vitro+CD8++T-lymphocyte+aging+and+may+participate+in+induced+death+sensitization+of+senescent+cells.">Autolysosomes accumulate during in vitro CD8+ T-lymphocyte aging and may participate in induced death sensitization of senescent cells.</a> Exp Gerontol. 39 (5), 789-800 (2004).</li><li>Poon, H. F., Vaishnav, R. A., Getchell, T. V., Getchell, M. L., Butterfield, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+proteomics+analysis+of+differential+protein+expression+and+oxidative+modification+of+specific+proteins+in+the+brains+of+old+mice.">Quantitative proteomics analysis of differential protein expression and oxidative modification of specific proteins in the brains of old mice.</a> Neurobiol Aging. 27 (7), 1010-1019 (2006).</li><li>Yin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+basis+of+lipofuscin,+ceroid,+and+age+pigment-like+fluorophores.">Biochemical basis of lipofuscin, ceroid, and age pigment-like fluorophores.</a> Free Radic Biol Med. 21 (6), 871-888 (1996).</li><li>Nehrke, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reduction+in+intestinal+cell+pHi+due+to+loss+of+the+Caenorhabditis+elegans+Na+/H++exchanger+NHX-2+increases+life+span.">A reduction in intestinal cell pHi due to loss of the Caenorhabditis elegans Na+/H+ exchanger NHX-2 increases life span.</a> J Biol Chem. 278 (45), 44657-44666 (2003).</li><li>Chakraborty, K., Leung, K., Krishnan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+lumenal+chloride+in+the+lysosome+is+critical+for+lysosome+function.">High lumenal chloride in the lysosome is critical for lysosome function.</a> Elife. 6, (2017).</li></ol>

Et utvalg av mobilnettet og molekylære hendelser bidra til aldring, påvirket av livshistorie trekk og genetiske faktorer. Våre siste studie22 antyder at den reproduktive syklusen spiller en viktig rolle i å kontrollere egnethet av soma gjennom regulering av lysosomale pH dynamics. Vi viste at lysosomale-mediert proteolyse flyttes mens dyr aktivt reprodusere av oppregulering av v-ATPase transkripsjon, som igjen sikrer surt lysosomer. På slutten av reproduksjon, v-ATPase uttrykk dråper, lysoso...

Utseendet på protein aggregater er allment akseptert for å være et kjennetegn på aldring i eukaryote celler1,2,3, og dannelsen av som antas å være blant prinsippet driverne av mobilnettet senescence4 , 5 , 6 , 7. det er økende bevis for at som celler alder, protein katabolisme er svekket, fører til en økning i protein aggregering. Sammenbruddet av proteolyse i aldring celler innebærer en svekkelse av autophagy8 , samt proteasome-mediert protein fornedrelse9. Til slutt, irreversible protein oksidering øker i gamle celler, ytterligere svekke protein katabolisme10.
Autophagy var i utgangspunktet tenkt å være en ikke-selektive prosess for bulk nedbrytning av skadet proteiner, men nyere studier har indikert at autophagy er svært selektive til katabolisme av protein aggregater og dysfunksjonelle organelles som ikke mottakelig for degradering via andre protein klaring mekanismer11. Under prosessen med autophagy, er skadet og aggregert proteiner sequestered i en dobbel-membran vesicle kalte autophagosome. Denne autophagosome deretter sikringer med surt organeller kalt lysosomer, som fører til forringelse av autophagosome Last12. Lysosomer representerer slutten-punktet i autophagic veien og delta i ulike cellulære prosesser som membran reparasjon, transcriptional kontroll og næringsstoffer sensing; utheving deres sentralisert rolle i mobilnettet homeostase (omtalt i ref. 13). Flere studier har vist en tilknytning mellom en alder-avhengige nedgang i lysosomale funksjon og forskjellige nevrodegenerative lidelser13. Konsekvent, kan gjenopprette lysosomale funksjon i eldre celler forsinke utbruddet av aldring-relaterte fenotyper14,15. Studier av sammensetningen av intralumen miljøet foreslår at sammenbruddet av lysosomale funksjon i eldre celler ikke er på grunn av en reduksjon i produksjonen av lysosomale proteaser16. Eventuelt har det blitt foreslått at tap av intralysosomal syre, en betenkelig behov av sin enzymatisk aktivitet, kanskje ligger til grunn fall i lysosome-mediert proteolyse17. For å kunne utforske denne hypotesen, er det viktig å utvikle reagenser og protokoller å undersøke dynamiske endringer i lysosomale pH i levende celler i en repliserbar og konsekvent måte.
Tarmen C. elegans er store metabolske vevet i worms, og det er en viktig regulator av systemisk homeostase og levetid. Vi har utviklet analyser for å vurdere endringer i surheten av lumen intestinal lysosomer ormer å bestemme hvordan lysosome-mediert proteolyse bidrar til aldring. Men pH-sensitive fluorophores er brukt tidligere i C. elegans merke intestinal lysosomer, ikke har det ennå vært et forsøk på å etablere en vellykket protokoll som kan oppdage små økninger i lysosomale pH i vivo18. Her gir vi en protokoll som kan brukes til å oppdage tap av lysosomale Surhet i intestinal cellene C. elegans med en enkel og praktisk fôring protokoll som inkorporerer en pH-sensitive fluorophore (cDCFDA) i OP50 mat.

<table border="0" cellpadding="0" cellspacing="0" width="1166">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:475px;">OP50 (E. coli)</td>
			<td style="width:188px;">Caenorhabditis Genetics Center</td>
			<td style="width:119px;"></td>
			<td style="width:385px;">Order online at https://cgc.umn.edu/strain/OP50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">5(6)-carboxy-2&#8217;,7&#8217;-dichlorofluorescein diacetate&#160;</td>
			<td style="width:188px;">ThermoFisher</td>
			<td style="width:119px;">C369</td>
			<td>Commonly known as cDCFDA</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:475px;">9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1&#39;H-2,2&#39;-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron</td>
			<td style="width:188px;">ThermoFisher</td>
			<td style="width:119px;">L7528</td>
			<td>Commonly known as Lysotracker Red</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Confocal microscope (e.g. Zeiss LSM 510)</td>
			<td style="width:188px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">ImageJ</td>
			<td style="width:188px;"></td>
			<td style="width:119px;"></td>
			<td>Download for free from https://imagej.nih.gov/ij/download.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">LB Broth powder</td>
			<td style="width:188px;">ThermoFisher</td>
			<td style="width:119px;">22700041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Bacto Agar</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">A5306-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">NaCl</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">S9888</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Bacto Peptone</td>
			<td style="width:188px;">Fisher Scientific</td>
			<td style="width:119px;">S71604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Cholesterol powder</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">C3045&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">CaCl2</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">449709</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">MgSO4</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">M7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">K3PO4</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">P5629</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Sodium Azide</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">S2002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">DMSO</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">D8418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Microscope Slides</td>
			<td style="width:188px;">VWR</td>
			<td style="width:119px;">48311-703</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Cover Slips</td>
			<td style="width:188px;">ThermoFisher</td>
			<td style="width:119px;">3406</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Agarose</td>
			<td style="width:188px;">Sigma</td>
			<td style="width:119px;">A6013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Incubator</td>
			<td style="width:188px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:475px;">Mirror or other smooth flat surface</td>
			<td style="width:188px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing lysosomal alkalinization in the intestine of live caenorhabditis elegans

Rundormer Caenorhabditis elegans (C. elegans) er en modell som er mye brukt levetid og utviklingsmessige trasé. Slike studier er tilrettelagt av gjennomsiktigheten av dyret, muligheten til å vanlige og motsatte genetiske analyser, det er relative enkelt generere fluorescently merket proteiner og bruk av fluorescerende fargestoffer som kan enten være microinjected i tidlig embryo eller inn sin mat (E. coli belastning OP50) merke mobilnettet organeller (f.eks 9-diethylamino-5 H-benzo (a) phenoxazine-5-en og (3-{2-[(1H,1'H-2,2'-bipyrrol-5-yl-kappaN(1)) methylidene]-2 H-pyrrol-5-yl-kappaN} - N - [2-(dimethylamino)ethyl]propanamidato)(difluoro)boron). Her presenterer vi bruk av et fluorescerende pH-sensitive fargestoff som flekker intestinal lysosomer, gir en visuell presentasjon av dynamisk, fysiologiske endringer i lysosomale surhetsgraden i live ormer. Denne protokollen måle ikke lysosomale pH, men heller tar sikte på å etablere en pålitelig metode for å vurdere fysiologiske relevante varianter i lysosomale surhet. cDCFDA er en celle-permeant stoff som er konvertert til den fluorescerende fluorophore 5-(and-6)-carboxy-2',7'-dichlorofluorescein (cDCF) ved hydrolyse av intracellulær esterases. Protonation innenfor lysosomer feller cDCF i disse organeller, hvor det akkumuleres. På grunn av sin lave pKa på 4,8, har dette fargestoffet blitt brukt som en pH sensor i gjær. Her beskriver vi bruk av cDCFDA som et supplement til mat å vurdere surheten av intestinal lysosomer i C. elegans. Denne teknikken tillater påvisning av alkalinizing lysosomer i levende dyr, og har en rekke eksperimentelle programmer inkludert studier på aldring, autophagy og lysosomale biogenesis.

cDCFDA flekker lysosomer på en pH-avhengig måte, og dens lav pKa og klar opptak i lysosomer gjør det ideelle pH sensor21. cDCFDA flekker intensitet er omvendt proporsjonal med lysosomale pH (dvs. farging intensitet øker som pH reduseres)18,22. cDCFDA signaler er konsekvent svake lysosomer dyr behandlet med 20 mM chloroquine, en inhibitor av lysosomale forsuring og ormer utarmet av v-ATPase, pro...

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Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

Trinnvise instruksjoner å undersøke tap av lysosomale surhetsgraden i tarmen, C. elegans med pH-sensitive viktig fargestoff 5 (6) - carboxy - 2', 7'-dichlorofluorescein diacetate (cDCFDA)

Vurdere lysosomale Alkalinization i tarmen Live Caenorhabditis elegans

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are ...

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Developmental Biology

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Helhetlig vurdering av kimbane Chemical Toxicity Bruke Nematode<em&gt; Caenorhabditis elegans</em

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

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The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

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In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

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The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

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C. elegans tarmen som en modell for intercellulære Lumen Morphogenesis og I Vivo polarisert membran Biogenesis på encellede nivå: merking av antistoff flekker, RNAi tap av funksjon analyse og bildebehandling

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the ...

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

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Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Bruk av RNAi og varme-sjokk-indusert transkripsjon faktor uttrykk å omprogrammere bakterie celler til nerveceller i C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Beregningsorientert analyse av Caenorhabditis elegans Germline å studere distribusjon av kjerner, proteiner og Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

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Studere oksidativt Stress forårsaket av Mitis gruppe Streptococci i Caenorhabditis elegans

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented ...

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Isotropic lys-ark mikroskopi og automatisert Cell Lineage analyser til katalog Caenorhabditis elegans embryogenesis med Subcellulære oppløsning

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...