Name: passaging hues human embryonic stem cell-lines with trypsin.
Uploaded: 2006-10-12T00:00:00
Description: Watch this Scientific Journal Video about Passaging HuES Human Embryonic Stem Cell-lines with Trypsin. at JoVE.com

Generelt Vi anbefaler tining ikke mer enn én prøve på et gitt tidspunkt for å sikre enkel håndtering. Behandleren bør vokte seg for å forlate kulturer ved romtemperatur og lave CO2 i lange perioder av gangen. Alle sentrifugering av levende celler er gjort på 500-600 xg i 5 min ved romtemperatur. MEFs (Mus Embryonale Feeder Cells) <ol><li> Pre-varme MEF media til 37 ° C. Fjern MEF hetteglass fra -80 ° C og umiddelbart senk den nederste halvdelen av røre...

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	<li>Edmond, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nosocomial+bloodstream+infections+in+United+States+hospitals:+a+three-year+analysis.">Nosocomial bloodstream infections in United States hospitals: a three-year analysis.</a> Clin. Infect. Dis. 29, 239-244 (1999).</li><li>Ramage, G., Bachmann, S., Patterson, T. F., Wickes, B. L., Lopez-Ribot, 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=Investigation+of+multidrug+efflux+pumps+in+relation+to+fluconazole+resistance+in+Candida+albicans+biofilms.">Investigation of multidrug efflux pumps in relation to fluconazole resistance in Candida albicans biofilms.</a> J. Antimicrob. Chemother. 49, 973-980 (2002).</li><li>Srinivasan, A., Uppuluri, P., Lopez-Ribot, J., Ramasubramanian, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+High-Throughput+Candida+albicans+Biofilm+Chip.">Development of a High-Throughput Candida albicans Biofilm Chip.</a> PLoS ONE. 6, 19036-19036 (2011).</li><li>Ramage, G., Vandewalle, K., Wickes, B. L., Lopez-Ribot, 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=Characteristics+of+biofilm+formation+by+Candida+albicans.">Characteristics of biofilm formation by Candida albicans.</a> Rev. Iberoam. Micol. 18, 163-170 (2001).</li><li>Pierce, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+reproducible+96-well+plate-based+method+for+the+formation+of+fungal+biofilms+and+its+application+to+antifungal+susceptibility+testing.">A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing.</a> Nat. Protoc. 3, 1494-1500 (2008).</li><li>Lee, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cellular+microarray+for+high-throughput+toxicology+assays.">Three-dimensional cellular microarray for high-throughput toxicology assays.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 59-63 (2008).</li><li>Meyerhofer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+resist+films+produced+by+spinning.">Characteristics of resist films produced by spinning.</a> Journal of Applied Physics. 49, (1978).</li><li>Ramage, G., Vande Walle, K., Wickes, B. L., Lopez-Ribot, 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=Standardized+method+for+in+vitro+antifungal+susceptibility+testing+of+Candida+albicans+biofilms.">Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms.</a> Antimicrob. Agents Chemother. 45, 2475-2479 (2001).</li><li>Chandra, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+formation+by+the+fungal+pathogen+Candida+albicans:+Development,+architecture,+and+drug+resistance.">Biofilm formation by the fungal pathogen Candida albicans: Development, architecture, and drug resistance.</a> Journal of Bacteriology. 183, 5385-5394 (2001).</li><li>Jabra-Rizk, M. A., Falkler, W. A., Meiller, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fungal+biofilms+and+drug+resistance.">Fungal biofilms and drug resistance.</a> Emerg. Infect. Dis. 10, 14-19 (2004).</li><li>Tobudic, S., Lassnigg, A., Kratzer, C., Graninger, W., Presterl, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+activity+of+amphotericin+B,+caspofungin+and+posaconazole+on+Candida+albicans+biofilms+in+intermediate+and+mature+development+phases.">Antifungal activity of amphotericin B, caspofungin and posaconazole on Candida albicans biofilms in intermediate and mature development phases.</a> Mycoses. 53, 208-214 (2010).</li></ol>

I denne videoen viser vi hvordan vi rutinemessig passasje fargetoner humane embryonale stamcellelinjer i vårt laboratorium. Effektiv og regelmessig splitting og passaging er avgjørende for å opprettholde en sunn humane embryonale stamceller linje. Trypsin mediert passasje er en betydelig fordel av fargetoner cellelinjer, er det imidlertid viktig å ikke over trypsinize kulturer eller å ha en stor andel av enkeltceller under re-platting.

Hues media 651,5 ml KO-DMEM 500 ml PenStrep 6,5 ml GlutaMAXTM 6,5 ml NEAA 6,5 ml 2-mercaptoethanol 0.5ul KO Serum Replacement 65 ml Plasmanate 65 ml bFGF2 (10 ng / ml endelig) 0,65 ml

passaging hues human embryonic stem cell-lines with trypsin.

I denne videoen viser vi hvordan vår lab rutinemessig luftveiene fargetoner humane embryonale stamcellelinjer med trypsin. Humane embryonale stamceller er gjenstander av cellekultur, og har en tendens til å erverve karyotypic unormalt med høy befolkningstetthet doblinger. Riktig passaging er avgjørende for å opprettholde en sunn, udifferensiert, karyotypically normal Hues menneskelige embryonale stamceller kultur. Først blir en ekspanderende kultur vasket i PBS for å fjerne rester av media og celleavfall, da cellene er kledde med en minimal mengde varme 0,05% Trypsin-EDTA. Trypsin er igjen på cellene for opp til fem minutter, da cellene er forsiktig forskyves med en 2ml serologisk pipette. Cellesuspensjonen samles og blandes med et stort volum av fargetoner media, blir deretter cellene samles ved forsiktig sentrifugering. Den inaktiverte trypsin media blanding fjernes, og cellene resuspendert i forvarmet fargetoner media. En passende Delingsforholdet beregnes (vanligvis 01:10-01:20), og celler re-belagt på en 1-2 dagers gammelplate som inneholder en monolayer av bestrålte mus embryonale fibroblast mater celler. Den nylig sådd Hues kultur plate er igjen urørt i 48 timer, så media blir endret hver dag etterpå. Det er viktig å ikke trpsinize ned til en enkelt celle suspensjon, da dette øker risikoen for å innføre karyotypic misdannelser.

Watch this Scientific Journal Video about Passaging HuES Human Embryonic Stem Cell-lines with Trypsin. at JoVE.com

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

I denne videoen viser vi hvordan vår lab rutinemessig luftveiene fargetoner humane embryonale stamcellelinjer med trypsin.

Passaging Hues humane embryonale stamcelle-linjer med Trypsin

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

Research

JoVE Journal

Biology

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

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Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

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Chromatin Immunoprecipitation from Human Embryonic Stem Cells

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Culture and Maintenance of Human Embryonic Stem Cells  

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Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

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Freezing and Thawing Human Embryonic Stem Cells

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Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...