Name: detection of mitochondria membrane potential to study clic4 knockdown-induced hn4 cell apoptosis in vitro
Uploaded: 2018-07-17T13:00:00
Description: Watch this Scientific Journal Video about Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro at JoVE.com

Vi takker Mr. Chao Fang for cellekultur. Dette arbeidet ble støttet av tilskudd fra Natural Science Foundation av Kina (Grant nr. 81570403, 81371284); Anhui provinsielle Natural Science Foundation (Grant No. 1408085MH158); Fremragende unge Investigator Anhui medisinske universitet; Støtter programmet for utmerket unge talenter i universitetene i provinsen Anhui.

1. celle kultur og hva
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Cellekultur Merk: HN4, en HNSCC celle linje, ble avledet fra pasienter med HNSCC14.<ol>
<li>Kultur HN4 celler i Dulbeccos endret Eagle medium (DMEM, 4,5 finans glukose) med 10% fosterets bovin serum og antibiotika (100 U/mL penicillin og 100 µg/mL streptomycin). Inkuber cellene på 37 ° C med 5% CO2.</li>
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Cellen transfection
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<li>En dag før transfection, plate 5 x 105 celler i 2 ...

<ol>
	<li>Chen, J., Liao, W., Gao, W., Huang, J., Gao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermittent+hypoxia+protects+cerebral+mitochondrial+function+from+calcium+overload.">Intermittent hypoxia protects cerebral mitochondrial function from calcium overload.</a> Acta Neurol Belg. 113, 507-513 (2013).</li><li>Gogol, P., Trzci&#324;ska, M., Bry&#322;a, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motility,+mitochondrial+membrane+potential+and+ATP+content+of+rabbit+spermatozoa+stored+in+extender+supplemented+with+GnRH+analogue+[des-Gly10,+D-Ala6]-LH-RH+ethylamide.">Motility, mitochondrial membrane potential and ATP content of rabbit spermatozoa stored in extender supplemented with GnRH analogue [des-Gly10, D-Ala6]-LH-RH ethylamide.</a> Pol J Vet Sci. 17, (2014).</li><li>Machiels, 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=Advances+in+the+management+of+squamous+cell+carcinoma+of+the+head+and+neck.">Advances in the management of squamous cell carcinoma of the head and neck.</a> F1000Prime Rep. 6, 44 (2014).</li><li>Behera, 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=Concurrent+therapy+with+taxane+versus+non-taxane+containing+regimens+in+locally+advanced+squamous+cell+carcinomas+of+the+head+and+neck+(SCCHN):+a+systematic+review.">Concurrent therapy with taxane versus non-taxane containing regimens in locally advanced squamous cell carcinomas of the head and neck (SCCHN): a systematic review.</a> Oral Oncol. 50, 888-894 (2014).</li><li>Pancari, P., Mehra, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+therapy+for+squamous+cell+carcinoma+of+the+head+and+neck.">Systemic therapy for squamous cell carcinoma of the head and neck.</a> Surgical Oncology Clinics of North America. 24, 437-454 (2015).</li><li>Peretti, 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=Chloride+channels+in+cancer:+Focus+on+chloride+intracellular+channel+1+and+4+(CLIC1+AND+CLIC4)+proteins+in+tumor+development+and+as+novel+therapeutic+targets.">Chloride channels in cancer: Focus on chloride intracellular channel 1 and 4 (CLIC1 AND CLIC4) proteins in tumor development and as novel therapeutic targets.</a> Biochim Biophys Acta. 1848, 2523-2531 (2015).</li><li>Jentsch, T. J., Stein, V., Weinreich, F., Zdebik, A. 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S., Mutoh, M., Gerdes, M., Yuspa, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLIC4,+an+intracellular+chloride+channel+protein,+is+a+novel+molecular+target+for+cancer+therapy.">CLIC4, an intracellular chloride channel protein, is a novel molecular target for cancer therapy.</a> J Invest Derm Symp P. 10, 105-109 (2005).</li><li>Zhong, J., Kong, X., Zhang, H., Yu, C., Xu, Y., Kang, J., Yu, H., Yi, H., Yang, X., Sun, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+CLIC4+Enhances+Autophagy+and+Triggers+Mitochondrial+and+ER+Stress-Induced+Apoptosis+in+Human+Glioma+U251+Cells+under+Starvation.">Inhibition of CLIC4 Enhances Autophagy and Triggers Mitochondrial and ER Stress-Induced Apoptosis in Human Glioma U251 Cells under Starvation.</a> PloS one. 7, 39378 (2012).</li><li>Ponnalagu, D., Singh, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anion+Channels+of+Mitochondria.">Anion Channels of Mitochondria.</a> Handbook of Experimental Pharmacology. , (2016).</li><li>Leanza, L., 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=Intracellular+ion+channels+and+cancer.">Intracellular ion channels and cancer.</a> Front Physiol. 4, 227 (2013).</li><li>Kim, S. Y., Chu, K. C., Lee, H. R., Lee, K. S., Carey, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+nine+new+head+and+neck+cancer+cell+lines.">Establishment and characterization of nine new head and neck cancer cell lines.</a> Acta Oto-Laryngologica. 117, 775-784 (1997).</li><li>Jia, L., Liu, W., Guan, L., Lu, M., Wang, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Calcium-Activated+Chloride+Channel+ANO1/TMEM16A+Suppresses+Tumor+Growth+and+Invasion+in+Human+Lung+Cancer.">Inhibition of Calcium-Activated Chloride Channel ANO1/TMEM16A Suppresses Tumor Growth and Invasion in Human Lung Cancer.</a> PloS one. 10, 0136584 (2015).</li><li>Xu, 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=Suppression+of+CLIC4/mtCLIC+enhances+hydrogen+peroxide-induced+apoptosis+in+C6+glioma+cells.">Suppression of CLIC4/mtCLIC enhances hydrogen peroxide-induced apoptosis in C6 glioma cells.</a> Oncol Rep. 29, 1483-1491 (2013).</li><li>Zhu, W., Wang, X., Zhou, Y., Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C2-ceramide+induces+cell+death+and+protective+autophagy+in+head+and+neck+squamous+cell+carcinoma+cells.">C2-ceramide induces cell death and protective autophagy in head and neck squamous cell carcinoma cells.</a> Int J Mol Sci. 15, 3336-3355 (2014).</li><li>McFarland, R., Taylor, R. 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Det er godt dokumentert at Cl− kanaler er avgjørende i å opprettholde hemostasen av interne miljøet og spille viktige roller i celle spredning og apoptose15,16. Derfor forstå forholdet mellom ion kanal-målrettet intervensjon og apoptose er stort behov og betydning for å finne en bedre terapeutisk tilnærming for ulike kreftformer17. Mitokondrier opprettholde normale biologiske staten i en celle, og dens funksjon gjelder ...

Rh123 er et kationisk fluorescens fargestoff, som fungerer som en indikator for transmembrane potensial. Rh123 er dugelig av gjennomtrengende cellemembranen og angi mitokondrie matrix avhengig av potensielle forskjellen på innsiden og utsiden av membran1. Apoptose fører til skade mitokondrie membran integritet. Mitokondrie permeabiliteten overgangen pore (MPTP) åpnes og føre til kollaps av MMP, som igjen fører til utgivelsen av Rh123 på utsiden av mitokondrier. Til slutt, grønne fluorescens sterke vil bli oppdaget under fluorescens mikroskop. Det er godt dokumentert at uttømming av MMP og forhøyet membran permeabilitet er tidlige tegn på celle apoptose2. Rh123 kan derfor brukes til gjenkjenning av MMP endring og forekomsten av celle apoptose.
Som de 6 vanligste carcinoma i verden forverres hode og nakke kreft alvorlig en persons helse3. Selv om mange tilnærminger ble utviklet i de senere årene, er kliniske utfallet av behandling for pasienter som lider av hode og nakke squamous celle carcinoma (HNSCC) fremdeles ikke ideelt4. Utforske nye terapeutiske metoder kan forbedre behandling for HNSCC5. Ionekanaler som involverer mange biologiske prosesser viser en viktig rolle i utviklingen av ulike kreftformer6. Delvis eller totalt deltakelse av Cl- -kanaler er sterkt involvert i ulike egenskaper for neoplastic transformasjon inkludert aktive migrasjon, høy grad av spredning og invasiveness. I lys av dette, har CLIC, en roman protein familie, vært listet som en lovende klasse av terapeutiske mål for kreft behandling6,7. Nyere undersøkelser avdekket som familiemedlemmer CLIC inkludert CLIC1, CLIC4 og CLIC5, lokalisere til hjerte mitokondrie og reaktive oksygen arter (ROS) er upregulated av CLIC5, som angir funksjonell rolle mitokondrie ligger Cl - kanaler i apoptotisk svar8. CLIC4, en familiemedlemmer CLIC (også kjent som mtCLIC, P64H1 og RS43), har blitt mest grundig undersøkt for egenskapene apoptotisk regulering kreftceller og subcellular plassering inkludert Golgi, endoplasmatiske retikulum og mitokondrie i menneskelig keratinocytter7,9,10. Profilen for expression av CLIC4 var regulert av tumor nekrose faktor-α (TNF-α), P53 og ekstern stimulans. Overuttrykte og downregulation av CLIC4 utløse en apoptotisk respons hovedsakelig gjennom mitokondrie veien sammen med ubalansen av Bcl-2 familiemedlemmer, aktivering av caspase gjennomgripende og utgivelsen av cytochrome C11, 12 , 13. derfor MMP måling er avgjørende å utforske CLIC4-relaterte apoptose, og Rh123 fungerer som en ideell fluorescens indikator.
Studien beskriver en detaljert protokoll for påvisning av MMP å studere CLIC4 knockdown-indusert apoptose i HN4 celler. Rh123 brukes som en fluorescens sonde for å observere endringen av MMP. Under felles fluorescens mikroskop og AC confocal laser skanning fluorescens mikroskop, kan sanntid svingninger i MMP være løst. Vi diskuterer fordeler og begrensninger av AC confocal laserskanning fluorescens mikroskopet i detalj, og også sammenligne den med andre metoder. Denne protokollen kan også brukes til andre apoptose-relaterte studier.

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	<colgroup>
		<col />
		<col span="2" />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:372px;">HNSCC cells</td>
			<td style="width:183px;">ATCC</td>
			<td style="width:183px;">CRL-3241</td>
			<td style="width:559px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Polylysine</td>
			<td>Thermo Fisher Scientific</td>
			<td>P4981</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Specific siRNA for human CLIC4</td>
			<td>Biomics</td>
			<td>NM_013943</td>
			<td style="width:559px;">(accession numbers, NM_013943; corresponding to the cDNA sequence 
			5-GCTGAAGGAGGAGGACAAAGA-3) and scrambled siRNA (5 ACGCGUAACGCGGGAAUUU-3) were designed and obtained from Biomics Company</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Lipofectamine 2000 Transfection Reagent&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>L3000-015</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Opti-MEM I Reduced Serum Medium, GlutaMAX</td>
			<td>Thermo Fisher Scientific</td>
			<td>51985-042</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Rhodamine 123, FluoroPure grede</td>
			<td>Thermo Fisher Scientific</td>
			<td>R22420</td>
			<td></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:372px;">Dulbecco&#8217;smodified Eagle medium (DMEM, 4.5 g/L glucose)</td>
			<td>Gibco</td>
			<td>11965-084</td>
			<td style="width:559px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:372px;">Fetal Bovine Serum, Qualified, Australia Origin</td>
			<td>Gibco</td>
			<td>10099141</td>
			<td style="width:559px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:372px;">Trypsin-EDTA Solution</td>
			<td>Beyotime</td>
			<td>C0201</td>
			<td style="width:559px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:372px;">Antibiotic-Antimycotic, 100X</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td style="width:559px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Laser Scanning Confocal Microscopy</td>
			<td>Leica Microsystems GmbH</td>
			<td>LEICA.SP5-DMI6000-DIC</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Nikon Eclipse TE300 Inverted Microscope</td>
			<td>Nikon</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Metaflour, V7.5.0.0</td>
			<td>Universal Imaging Corporation</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Leica application suite, v2.6.0.7266</td>
			<td>Leica Microsystems GmbH</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microsoft office Excel 2007</td>
			<td>Microsoft</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sigma Plot 12.5</td>
			<td>Systat Software</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Attofluor Cell Chamber</td>
			<td>Thermo Fisher Scientific</td>
			<td>A7816</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of mitochondria membrane potential to study clic4 knockdown-induced hn4 cell apoptosis in vitro

Utarming av mitokondrie membranen potensielle (MMP, ΔΨm) regnes tidligste hendelsen i apoptotisk kaskade. Det skjer selv før kjernefysiske apoptotisk egenskaper, inkludert chromatin kondens og DNA brekkasje. Når MMP kollapser, celle apoptose starte irreversibelt. En rekke lipofile kationisk fargestoffer kan passere gjennom cellemembranen samlet i matrisen mitokondrie og tjene som fluorescens markør å vurdere MMP endring. Som en av seks medlemmene av Cl- intracellulær kanal (CLIC) familie deltar CLIC4 i celle apoptotisk prosessen hovedsakelig gjennom mitokondrie sti. Her beskriver vi en detaljert protokoll for å måle MMP via overvåking fluorescens svingninger i Rhodamine 123 (Rh123), hvor vi studere apoptose indusert av CLIC4 knockdown. Vi diskuterer fordeler og begrensninger av AC confocal laserskanning og normal fluorescens mikroskopet i detalj, og også sammenligne den med andre metoder.

Studien, ble Rh123 brukt for å oppdage MMP. Først ble HN4 celler kultivert til følgende fluorescens flekker eksperimenter. Pinsett ble brukt til å sette sirkulære coverslips på bunnen av 6-vel plater (figur 1A). Coverslips ble belagt med polylysine i 5 minutter og deretter polylysine fjernet via hindre (figur 1B). Deretter var HN4 celler trypsinized og sådd på coverslips plassert i bunnen...

Watch this Scientific Journal Video about Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro at JoVE.com

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Her presenterer vi en detaljert protokoll for anvendelse av rhodamine 123 å identifisere mitokondrie membranen potensielle (MMP) og studere CLIC4 knockdown-indusert HN4 celle apoptose i vitro. Under felles fluorescens mikroskop og AC confocal laser skanning fluorescens mikroskop, ble real-time endringen av MMP registrert.

Påvisning av mitokondrier membran potensial til å studere CLIC4 Knockdown-indusert HN4 celle apoptose In Vitro

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of ...

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Cancer Research

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

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Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

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Sanntid påvisning av i Vitro svulst celle apoptose indusert av CD8+ T celler å studere immun undertrykkende funksjoner av svulst-infiltrere myeloide celler

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Overføring av manipulert tumor-Associated nøytrofile til tumor-bærende mus for å studere deres angiogenic potensial in vivo

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In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

In vivo Immunofluorescence lokalisering for vurdering av terapeutiske og diagnostiske antistoff Biodistribusjon i Cancer Research

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in ...

In Vitro Assay to Study Tumor-macrophage Interaction

In vitro-analysen for å studere tumor-macrophage interaksjon

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Cell Cycle-specific Measurement of &#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

Cell Cycle-specific Measurement of ÃƒÅ½Ã‚Â³H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

Celle syklus-spesifikk måling av γH2AX og apoptose etter gentoksisk stress av Flow flowcytometri

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer ...

Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers

Bruke kylling chorioallantoic membran i Vivo modell for å studere gynekologiske og urologiske kreftformer

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo ...