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 vil gerne takke Mr. Chao Fang for cellekultur. Dette arbejde blev støttet af tilskud fra Natural Science Foundation of China (Grant No. 81570403, 81371284); Anhui Provincial Natural Science Foundation (Grant No. 1408085MH158); Fremragende unge Investigator i Anhui medicinske universitet; Understøtter Program for fremragende unge talenter i universiteter af Anhui-provinsen.

1. celle kultur og Transfektion
<ol><li>
Cellekultur Bemærk: HN4, en HNSCC cellelinie, stammer fra patienter med HNSCC14.<ol>
<li>Kultur HN4 celler i Dulbeccos ændrede Eagle medium (DMEM, 4,5 g/L glukose) suppleret med 10% føtal bovint serum og antibiotika (100 U/mL penicillin og 100 µg/mL streptomycin). Inkuber celler ved 37 ° C med 5% CO2.</li>
	</ol>
</li>
	<li>
Celle Transfektion
	<ol>
<li>En dag før Transfektion, plade 5 x 105...

<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 veldokumenteret, at Cl− kanaler er afgørende for opretholdelsen af hæmostase af det indre miljø og spiller en vigtig rolle i celledelingen og apoptose15,16. Derfor forstå forholdet mellem ion-kanal-målrettet intervention og apoptose er stort behov og betydning til at finde en bedre terapeutisk tilgang til forskellige kræftformer17. Mitokondrier opretholde normale biologiske tilstand af en celle og dens funktion ve...

Rh123 er en kationiske fluorescens dye, der tjener som en indikator for transmembrane potentiale. Rh123 er i stand til at trænge ind i cellemembranen og ind i mitokondriets matrix afhængigt af spændingsforskel på inde og udenfor membran1. Apoptose fører til beskadigelse af mitokondrielle membran integritet. Mitochondriet permeabilitet overgangen pore (MPTP) vil åbne og fører til kollaps af MMP, hvilket igen resulterer i frigivelse af Rh123 på ydersiden af mitokondrier. Endelig, stærkere grønne fluorescens signal registreres under fluorescens mikroskop. Det er veldokumenteret, at udtømning af MMP og forhøjede membran permeabilitet er tidlige tegn på celle apoptose2. Rh123 kan derfor anvendes til påvisning af MMP forandringer og forekomsten af celle apoptose.
Som de 6 mest almindelige karcinom i verden forringes hoved og hals kræft alvorligt en persons sundhed3. Selv om mange tilgange blev udviklet i de seneste år, er kliniske resultater af behandling af patienter med hoved og hals planocellulært karcinom (HNSCC) stadig ikke ideelle4. At udforske nye terapeutiske metoder kan forbedre behandlingen for HNSCC5. Ionkanaler involverer mange biologiske processer vise en vigtig rolle i udviklingen af forskellige kræftformer6. Hel eller delvis deltagelse af Cl- kanaler er stærkt involveret i forskellige egenskaber af neoplastiske transformation herunder aktive migration, høj rate af spredning og invasiv. I lyset af dette, har CLIC, en roman protein familie, været opført som en lovende klasse af terapeutiske mål for kræft behandling6,7. Nylige undersøgelser har vist, at medlemmer af familien CLIC, herunder CLIC1, CLIC4 og CLIC5, lokalisere til hjerte mitokondrie og reaktive ilt arter (ROS) niveau er upregulated af CLIC5, som angiver den funktionelle rolle af mitokondrie-beliggende Cl - kanaler i apoptotiske svar8. CLIC4, en medlemmer af familien CLIC (også kendt som mtCLIC, P64H1 og RS43), har været mest grundigt undersøgt for sin apoptotiske forordning egenskaber i kræftceller og subcellulært placering herunder Golgi, endoplasmatiske reticulum og mitochondriet i menneskelige keratinocytter7,9,10. Profilen udtryk af CLIC4 blev reguleret af tumor nekrose faktor-α (TNF-α), P53 og eksterne stimuli. Overekspression og downregulation af CLIC4 udløse en apoptotiske svar hovedsagelig gennem den mitokondrielle vej ledsaget med ubalance af Bcl-2 familiemedlemmer, aktivering af caspase cascade og frigivelse af cytokrom C11, 12 , 13. derfor MMP måling er afgørende at udforske CLIC4-relaterede apoptose, og Rh123 fungerer som en ideel fluorescens-indikator.
Den nuværende undersøgelse beskriver en detaljeret protokol til påvisning af MMP at studere CLIC4 knockdown-induceret apoptose i HN4 celler. Rh123 bruges som et fluorescens sonde til at observere ændringen af MMP. Under fælles fluorescens mikroskop og konfokal laser scanning fluorescens mikroskop, kan real-time udsving i MMP blive løst. Vi diskutere fordele og begrænsninger i anvendelsen af Konfokal laser scanning fluorescens mikroskop i detaljer, og også sammenligne det med andre metoder. Denne protokol kan også anvendes til andre apoptose-relaterede 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

Nedbrydningen af de mitokondrielle membran potentiale (MMP, ΔΨm) anses for den tidligste begivenhed i den apoptotiske kaskade. Det sker endda foran nukleare apoptotiske karakteristika, herunder kromatin kondens og DNA brud. Når MMP kollapser, celle apoptose vil indlede uigenkaldeligt. En serie af lipofile kationiske farvestoffer kan passere gennem cellemembranen og aggregere inde matrix af mitochondriet og tjene som fluorescens markør til at evaluere MMP ændring. Som en af seks medlemmer af Cl- intracellulære kanal (CLIC) familie, deltager CLIC4 i celle apoptotiske proces hovedsagelig gennem den mitokondrielle vej. Her beskriver vi en detaljeret protokol for at måle MMP via overvågning fluorescens udsving af rhodaminfilteret 123 (Rh123), hvorigennem vi studere apoptose induceret af CLIC4 knockdown. Vi diskutere fordele og begrænsninger i anvendelsen af Konfokal laser scanning og normal fluorescens mikroskop i detaljer, og også sammenligne det med andre metoder.

I den nuværende undersøgelse, blev Rh123 anvendes til påvisning af MMP. I første omgang, blev HN4 celler kulturperler for de følgende fluorescens farvning eksperimenter. Pincet blev brugt til at sætte cirkulære coverslips på bunden af 6-godt plader (fig. 1A). Coverslips var belagt med polylysine i 5 min og derefter polylysine fjernes via pipette (figur 1B). Derefter blev HN4 celler trypsi...

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 præsenterer vi en detaljeret protokol om anvendelse af rhodaminfilteret 123 til at identificere den mitokondrielle membran potentiale (MMP) og studere CLIC4 knockdown-induceret HN4 celle apoptose in vitro. Under fælles fluorescens mikroskop og konfokal laser scanning fluorescens mikroskop, blev real-time ændringen af MMP indspillet.

Påvisning af mitokondrier membran potentiale til at studere CLIC4 Knockdown-induceret 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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Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

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Realtid påvisning af i Vitro Tumor celle apoptose induceret af CD8+ T-celler til at studere immun suppressiv funktioner af Tumor infiltrerer myeloide celler

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Overførsel af manipulerede tumor-associerede neutrofiler til tumor bærende mus for at studere deres angiogene potentiale in vivo

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the ...

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

In vivo immunofluorescens lokalisering til vurdering af terapeutisk og diagnostisk antistof Biodistribution i kræftforskning

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-analyse for at studere tumor-makrofag interaktion

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 cyklus-specifik måling af γH2AX og apoptose efter genotoksisk stress ved flow cytometri

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

Brug af kylling chorioallantoic membran i Vivo Model til at studere gynækologiske og urologiske kræftformer

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 ...