Name: toxoplasma gondii cyst wall formation in activated bone marrow-derived macrophages and bradyzoite conditions
Uploaded: 2010-08-12T17:30:00
Description: Watch this Scientific Journal Video about Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions at JoVE.com

Detta arbete stöddes av University of Wisconsin-Madison Herman I. Shapiro Distinguished Graduate Fellowship (CMT), NIH utmärkelse AI072817 (AMP) och American Heart Association Award 0840059N (LJK).

1. Beredning av mänskliga förhuden fibroblaster (HFF)-belagda täckglas <ol><li> Placera en steril runda glas täckglas på botten av brunnarna i ett 24-och vävnadsodling plattan. </li><li> Att skörda HFFs från en konfluenta 150cm 2 kolv, skölj kolven två gånger med 1X PBS och tillsätt 2,5 ml 0,025% trypsin-EDTA. Inkubera kolv vid 37 ° C i 5-10 minuter. </li><li> Genom att peka på sidan av kolven mot handflatan kan stöd i loss celler från kolven. När celler har släppt från kolven, tillsätt ...

<ol>
	<li>Tomida, M., Yamamoto-Yamaguchi, Y., Hozumi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+a+factor+inducing+differentiation+of+mouse+myeloid+leukemic+M1+cells+from+conditioned+medium+of+mouse+fibroblast+L929+cells.">Purification of a factor inducing differentiation of mouse myeloid leukemic M1 cells from conditioned medium of mouse fibroblast L929 cells.</a> J Biol Chem. 259 (17), 10978-10980 (1984).</li><li>Matrajt, M., Donald, R. G., Singh, U., Roos, 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=Identification+and+characterization+of+differentiation+mutants+in+the+protozoan+parasite+Toxoplasma+gondii.">Identification and characterization of differentiation mutants in the protozoan parasite Toxoplasma gondii.</a> Mol Microbiol. 44, 735-747 (2002).</li><li>Singh, U., Brewer, J. L., Boothroyd, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+tachyzoite+to+bradyzoite+differentiation+mutants+in+Toxoplasma+gondii+reveals+a+hierarchy+of+gene+induction.">Genetic analysis of tachyzoite to bradyzoite differentiation mutants in Toxoplasma gondii reveals a hierarchy of gene induction.</a> Mol Microbiol. 44, 721-733 (2002).</li><li>J, P., Knoll, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Toxoplasma+gondii+development+mutants+identifies+a+potential+proteophosphogylcan+that+enhances+cyst+wall+formation.+Molecular+and+Biochemical+Parasitology.">Isolation of Toxoplasma gondii development mutants identifies a potential proteophosphogylcan that enhances cyst wall formation. Molecular and Biochemical Parasitology.</a> 169 (2), 120-123 (2010).</li><li>Bohne, W., Heesemann, J., Gross, U. <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+Bradyzoite-Specific+Toxoplasma+gondii+Antigens+in+Gamma+Interferon-Treated+Mouse+Macrophages.">Induction of Bradyzoite-Specific Toxoplasma gondii Antigens in Gamma Interferon-Treated Mouse Macrophages.</a> Infection and Immunity. 61 (3), 1141-1145 (1993).</li><li>Ibrahim, H. M., Bannai, H., Xuan, X., Nishikawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxoplasma+gondii+Cyclophilin+18-Mediated+Production+of+Nitric+Oxide+Induces+Bradyzoite+Conversion+in+a+CCR5-Dependent+Manner.">Toxoplasma gondii Cyclophilin 18-Mediated Production of Nitric Oxide Induces Bradyzoite Conversion in a CCR5-Dependent Manner.</a> Infection and Immunity. 77 (9), 3686-3695 (2009).</li><li>Lyons, R. E., McLeod, R., Roberts, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxoplasma+gondii+tachyzoite-bradyzoite+interconversion.">Toxoplasma gondii tachyzoite-bradyzoite interconversion.</a> Trends in Parasitology. 18 (5), 198-201 (2002).</li><li>Berghaus, L. J., Moore, J. N., Hurley, D. J., Vandenplas, M. L., Fortes, B. P., Wolfert, M. A., Boons, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+responses+of+primary+murine+macrophage-lineage+cells+and+RAW+264.7+cells+to+ligands+of+Toll-like+receptors+2,+3,+and+4.">Innate immune responses of primary murine macrophage-lineage cells and RAW 264.7 cells to ligands of Toll-like receptors 2, 3, and 4.</a> Comp Immunol Microbiol Infect Dis. , (2009).</li><li>Mordue, D. G., Scott-Weathers, C. F., Tobin, C. M., Knoll, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+patatin-like+protein+protects+Toxoplasma+gondii+from+degradation+in+activated+macrophages.">A patatin-like protein protects Toxoplasma gondii from degradation in activated macrophages.</a> Mol Microbiol. 63 (2), 482-496 (2007).</li><li>Bohne, W., Heesemann, J., Gross, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+replication+of+Toxoplasma+gondii+is+necessary+for+induction+of+bradyzoite-specific+antigens:+a+possible+role+for+nitric+oxide+in+triggering+stage+conversion.">Reduced replication of Toxoplasma gondii is necessary for induction of bradyzoite-specific antigens: a possible role for nitric oxide in triggering stage conversion.</a> Infection and Immunity. 62 (5), 1761-1777 (1994).</li></ol>

Trots att mekanismen för bradyzoite utvecklingen inte är helt klarlagda, molekylärgenetiska analyser av T. gondii skede konvertering i vävnadskultur har lett till upptäckten av gener som är inblandade i bradyzoite cystbildning 2,3,4. Analyserna ledde också till observationen att vissa bradyzoite markörer uttrycks i andra långvarig stress villkor, inklusive tillväxt i aktiverade makrofager 5,6. Ovanstående metoder beskriver hur man odlar T. gondii och framkalla utvecklin...

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<td>Bovine serum albumin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A7906</td>
<td>&nbsp;</td>
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<td>Dulbecco's Modified Eagle Medium (DMEM)</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11960-051</td>
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<td>Fetal Bovine Serum (FBS)</td>
<td>&nbsp;</td>
<td>Atlanta Biologicals</td>
<td>S11150</td>
<td>heat inactivate </td>
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<td>Rhodamine Dolichos biflorus agglutinin</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>RL-1032</td>
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<td>Polysciences</td>
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<td>BP381-5</td>
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<td>Fisher Scientific</td>
<td>BP310-1</td>
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<td>IFN&gamma;</td>
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<td>PeproTech</td>
<td>315-05</td>
<td>Store in single use aliquots</td>
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<td>L-glutamine (200mM)</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25030</td>
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<td>&nbsp;</td>
<td>Sigma</td>
<td>L4391-1MG</td>
<td>Store in single use aliquots</td>
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<td>Microscope Cover Glass</td>
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<td>Fisher Scientific</td>
<td>12-545-80 12CIR-1</td>
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<td>15140</td>
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<td>BP151-500</td>
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<td>Gibco</td>
<td>25200</td>
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<td>Vector Laboratories</td>
<td>H-1200</td>
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toxoplasma gondii cyst wall formation in activated bone marrow-derived macrophages and bradyzoite conditions

Toxoplasma gondii är en obligat intracellulär parasit som kan invadera alla kärnförsedda cell i varmblodiga djur. Under infektion, T. gondii sprider som en snabb replikering formulär som kallas tachyzoite. Tachyzoites konvertera till en långsamt växande encysted formen kallas bradyzoite genom ett signalsystem process som inte är väl karakteriserade. Inom djur, bradyzoite cystor finns i det centrala nervsystemet och muskelvävnad och representerar den kroniska skedet av infektionen. Konvertering till bradyzoites kan simuleras i vävnadskultur av CO 2 svält, med hjälp av medium med högt pH-värde, eller tillägg av interferon-gamma (IFNγ). Bradyzoites kännetecknas av närvaron av en cysta vägg, som lektin Dolichos biflorus agglutinin (DBA) binder. Fluorescerande DBA används för att visualisera cysta väggen i parasiter odlas i humana förhuden fibroblaster (HFFs) som har utsatts för låga CO 2 och högt pH medium. Likaså parasiter som bor i murina härledd från benmärg makrofager (BMMs) visa en cysta väggen upptäckas av DBA efter BMMs aktiveras med IFNγ och lipopolysackarid (LPS). Detta protokoll kommer att visa hur man kan få konvertering av T. gondii till bradyzoites med en hög medium pH tillväxt med låg CO 2 och aktivering av BMMs. Värdceller kommer att odlas på täckglas, infekterade med tachyzoites och antingen aktiveras med tillägg av IFNγ och LPS (BMMs) eller utsätts för högt pH tillväxt medium (HFFs) i tre dagar. Efter avslutad infektioner, kommer värdceller fastställas, permeabilized, och blockerade. Cysta väggar kommer att visualiseras med Rhodamine DBA med fluorescensmikroskopi.

Watch this Scientific Journal Video about Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions at JoVE.com

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii omvandlas till en cysta formulär som svar på miljöpåfrestningar, som kan härmade i modellerna vävnadsodling. Denna video visar tekniker för att undersöka cysta väggen bildas genom att aktivera härledd från benmärg makrofager eller ändrar pH-tillväxt medium fibroblast celler.

Toxoplasma gondii Cysta Wall Bildning i aktiverade härledd från benmärg makrofager och Bradyzoite Villkor

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii ...

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Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

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