Bu çalışma, Wisconsin-Madison Herman I. Shapiro Değerli Yüksek Lisans Bursu (CMT), NIH ödül AI072817 (AMP) ve Amerikan Kalp Derneği Ödülü 0840059N (LJK) Üniversitesi tarafından desteklenmiştir.

1. Insan sünnet derisi fibroblastlar (HFF) kaplı lamelleri hazırlanması <ol><li> 24-iyi bir doku kültürü plaka kuyulardan alt steril bir yuvarlak cam lamel yerleştirin. </li><li> Konfluent 150cm 2 balon paketleme hasat için 1X PBS ile şişeyi iki kez yıkayın ve 2.5 ml% 0.025 tripsin-EDTA ekleyin . 37 ° C&#39;de 5-10 dakika şişesi inkübe edin. </li><li> Elinizin avuç içi karşı balonun yan dokunulduğunda şişeden hücreler ayrılmakta yardımcı olabilir. Bir kez hücreleri şişeden piyasaya sahip, HFF orta 150 ml (Dulbecco&#39;nun Modifiye Kartal Orta []% 10 FBS ile DMEM, 2 mM L-glutamin, ve 1% penisilin-streptomisin) ve balondaki durulama ve toplamak için orta kullanmak eklemek hücreler. </li><li> Lamelleri ile başına hücre 1 ml koyun. </li><li> Hücreler 37 ° C,% 5 CO 2 konfluent olmak için izin verin. </li></ol> 2. L929 klimalı BMC gelişimi için orta (CM) hazırlanması <ol><li> L929 confluency uzun dönemlerinden sonra makrofaj koloni uyarıcı faktörü salgılar bir fare anöploid fibrosarkom hücre hattı. Bu salgı, fare kemik iliği hücrelerinin hücre kültürü 1 makrofajlar içine geliştirmek için kullanılacak L929 hücreleri CM sağlar . </li><li> L929 hücreleri konfluent sonra, onlar küresel görünür ve şişeyi kapalı kaldırma başlayana kadar onlara ek bir 7-9 gün inkübe edin. Süpernatant ve pelet, 10 dakika boyunca 425 xg&#39;de herhangi bir müstakil hücreleri toplayın. Bu süpernatant CM. </li><li> L929 hücreleri biri 150 cm 2 şişesi 150 ml BMC orta üretir CM 30 ml verir. BMC orta,% 10 FBS,% 20 CM DMEM içinde,% 1 penisilin-streptomisin, ve% 1 L-glutamin oluşur. </li></ol> 3. BMCs kemik iliği ve hücre kültürü izolasyonu <ol><li> CO 2 boğulma ve yüzey% 70 etanol ile sterilize bir C57BL / 6 fare Kurban. </li><li> Yakın karın cilt bacak kaldırma ve makasla kesme periton Açığa. Periton boşluğuna delinmesiyle kaçının. Tibia ve femur maruz bacak azaltın. Makas kullanarak bacak kemiklerinin kas dokusu kesin. </li><li> Femur kaldırmak için diz altında, bir kez ve bir kez kalça yakın kesti. Soğuk 1X PBS içeren bir bakteriyolojik Petri kabındaki kemiklerin yerleştirin. BMCs kalıcı uyacaktır çünkü bu adımlar için Petri kutularına tedavi doku kültürü kullanmayın. Ekstra kas dokusu kazımak için bir neşter kullanın. Sadece kemik iliği maruz diz üstünde kesin. </li><li> Adımları tekrarlayın (b) ve (c) ikinci femur kemik iliği toplamak için. </li><li> Soğuk 1X PBS ve 50 ml konik bir tüp içine kemik iliği çıkarmak için 25 gauge iğne ile dolu bir 10cc şırınga kullanın. Bembeyaz bir kez kemik iliği kemik kaldırılır görünmelidir. </li><li> PBS / kemik iliği karışımı, kemik iliği agrega kırmak için 22 gauge iğne ile iletin. </li><li> 10 dakika boyunca 425 xg&#39;de karışımı Spin, süpernatantı kaldırmak. </li><li> BMC orta 8 ml pelletini tekrar. 8 bakteriyolojik Petri kutularına 1 ml hücre süspansiyonu ve 9 ml BMC orta ekleyin. </li><li> 37 ° ° C&#39;de,% 5 CO 2. </li><li> 5 gün, her plaka için 10 ml BMC orta ekleyin. 7 gün, hücrelerin tam olarak gelişmiş olacak. BMCs olgunlaşma sonra 1-2 hafta geçişli olabilir. </li><li> BMCs bölmek için, orta kaldırmak ve her Petri kabı soğuk 1X PBS 5 ml ekleyin. 4 inkübe ° C, 30 dakika boyunca hücreleri kaldırmaya başlayana kadar. , Steril bir transfer pipet yardımıyla tabak kapatın BMCs durulayın. </li><li> 50 ml konik bir tüp ve pelet 10 dakika boyunca 425 xg&#39;de Havuz BMCs. </li><li> 10 ml BMC orta ve bir hemasitometre saymak pelletini tekrar. Tohum 2x105 altındaki yuvarlak cam lamelleri ile 24 plaka de ortalama BMCs. T. ile enfekte önce BMCs gecede uymak gondii. Fazla BMCs daha sonra kullanmak için Petri kutularına reseeded olabilir. </li></ol> 4. T. ile enfekte olan hücreleri gondii <ol><li> 2x10 6 T. ile 25 cm 2 konfluent paketleme balon enfekte gondii ve hücreler (yaklaşık 2-3 gün) lyse alıncaya kadar büyür. </li><li> Doku kültürü şişesi enfekte konak hücre tek tabaka kaldırmak için bir hücre kazıyıcı kullanın, daha sonra 27-gauge iğne ile yerinden tek tabaka geçen parazitler konak hücrelerine bırakın. </li><li> Orta parazitlerin sayısını belirlemek için bir hemasitometre kullanın. </li><li> Protokol 1 veya Protokolü 3 den BMCs HFF mono tabakaları lamel ile ortalama 10 5 parazitleri bulaştırabilir. Parazitler 37 az 3 saat boyunca işgal ° C,% 5 CO 2 olsun. </li></ol> 5. T. başlatılması çevresel stresler gondii bradyzoite geliştirme <ol><li> Orta pH artışı ve CO 2 açlık paketleme Bradyzoite geliştirme <ol class="lalpha"><li> Geliştirme ortamı hazırlayın. Kalkınma orta RPM içerirBen olmadan 1640 bikarbonat,% 1 FBS,% 1 penisilin-streptomisin ve 42 mM HEPES. pH 8.0 ve filtre sterilize edin. </li><li> Enfekte paketleme DMEM çıkarın, 1X PBS ile temizleyin, durulayın ve kalkınma orta 1ml eklemek. </li><li> Ortam havası ile 3 gün 37 ° ° C&#39;de inkübe edin. </li></ol></li><li> BMCs, aktivasyonu <ol class="lalpha"><li> BMCs etkinleştirmek orta hazırlayın. Etkinleştirme orta BMC orta 100U/ml IFN-γ ve 100 ng / ml LPS ile desteklenmiş. Agrega bozmak için kullanmadan önce 2 dakika süreyle, bir su banyosu LPS sonikasyon. </li><li> BMC orta kuyulardan çıkarın ve 1 ml aktivasyon orta ile değiştirin. </li><li> 37 ° ° C&#39;de,% 5 CO 2 3 gün boyunca. </li></ol></li></ol> 6. İmmünofloresan algılama <ol><li> 1X PBS ile enfekte kuyuları üç kez durulayın. </li><li> 20 dakika boyunca% 3 formaldehit 200ul ile tek tabaka sabitleyin. </li><li> Fiksatif çıkarın ve 1X PBS ile kuyu durulayın. </li><li> 0.1 M glisin 200μl kuyulara ekleyin ve 5 dakika bekletin. </li><li> Glisin çıkarın ve 1X PBS ile kuyu durulayın. </li><li> 30 dakika boyunca tek tabaka permeabilize ve blok 3% BSA/0.2% 1X PBS TritonX-100 250 ul ekleyin. </li><li> Engelleme çözüm çıkarın ve 1X PBS ile kuyu durulayın. </li><li> % 3 rodamin Dolichos tohumları biflorus agglutinin bir 1:250 dilüsyon 50 ul BSA/0.2% 1X PBS Triton X-100. </li><li> 1 saat oda sıcaklığında Kapak platformu çalkalayıcı üzerinde plaka ve yer </li><li> Her bir platform çalkalayıcı 5 dakika için 1X PBS Triton X-100,% 0.2 ile lamelleri 3 kere yıkayın. </li><li> 4&#39;6-diamidino-2-phenylindole (DAPI) içeren VectaShield montaj orta kullanarak Dağı lamelleri. </li><li> Vitray T. gondii 100x büyütme rodamin için uygun bir filtre ile floresan mikroskop kullanılarak görüntülendi olabilir. </li></ol> 7. Temsilcisi sonuçları Şekil 1 T. temsilcisi DBA boyanma gösterir pH stres altında aktive BMMs ve paketleme gondii. Parazit etrafında iki gösteri DBA boyama vakuollerde içeren kist duvar bileşenlerinin varlığını gösteren. Aktive BMM görüntü vakuol yüzeyi ile tutarlı DBA boyama gösterir. T. kesit gondii pH paketleme lekeli iç yapıları ile kist gösterir vurguladı. <img src="/files/ftp_upload/2091/2091fig1.jpg" alt="Şekil 1" /> Şekil 1. DBA boyama vurguladı T. stres koşullarına, aktif BMMs veya pH altında gondii. intrasellüler parazit, paketleme vurguladı rodamin konjuge DBA (kırmızı) ile boyandı. Diferansiyel girişim kontrast (DIC), kist anahat gösterir ve siyah ölçek çubuğu 2 mcM eşittir. Hayvanlar deneyler Hayvanlar üzerinde yapılan deneyler, University of Wisconsin Hayvan Bakım ve Kullanım Kurulu tarafından belirlenen kurallara ve yönetmeliklere uygun olarak yapıldı.

<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>

T. bradyzoite kalkınma mekanizması tam olarak anlaşılamamış olmakla birlikte, moleküler genetik analizler gondii doku kültüründe sahne dönüşüm bradyzoite kist oluşumu 2,3,4 yer alan genlerin keşfi yol açmıştır. Analizler de bradyzoite bazı işaretleri aktive makrofajlar 5,6 büyüme de dahil olmak üzere diğer uzun süreli stres koşullarına, ifade olduğunu gözleme açtı. Yukarıdaki yöntemlerden T. nasıl yetiştirileceğini tarif gondii</e...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Bovine serum albumin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A7906</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco's Modified Eagle Medium (DMEM)</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11960-051</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum (FBS)</td>
<td>&nbsp;</td>
<td>Atlanta Biologicals</td>
<td>S11150</td>
<td>heat inactivate </td>
</tr>
<tr>
<td>Rhodamine Dolichos biflorus agglutinin</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>RL-1032</td>
<td>&nbsp;</td>
<tr>
<td>Formaldehyde (16%)</td>
<td>&nbsp;</td>
<td>Polysciences</td>
<td>18814</td>
<td>&nbsp;</td>
<tr>
<td>Glycine</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP381-5</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP310-1</td>
<td>&nbsp;</td>
<tr>
<td>IFN&gamma;</td>
<td>&nbsp;</td>
<td>PeproTech</td>
<td>315-05</td>
<td>Store in single use aliquots</td>
</tr>
<tr>
<td>L-glutamine (200mM)</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Lipopolysaccharide (LPS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L4391-1MG</td>
<td>Store in single use aliquots</td>
</tr>
<tr>
<td>Microscope Cover Glass</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-545-80 12CIR-1</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>RPMI medium 1640 powder (with L-glutamine, without bicarbonate)</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>31800-022</td>
<td>&nbsp;</td>
<tr>
<td>Triton-X-100</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP151-500</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA (0.25%)</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25200</td>
<td>&nbsp;</td>
<tr>
<td>VectaShield mounting media with DAPI</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>H-1200</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

toxoplasma gondii cyst wall formation in activated bone marrow-derived macrophages and bradyzoite conditions

Toxoplasma gondii zorunlu hücre içi parazit, sıcak kanlı hayvanların herhangi bir çekirdekli hücre istila edebilir . Enfeksiyon sırasında, T. hızlı kopyalayan bir form tachyzoite denilen gondii yaymaktadır. Tachyzoites iyi karakterize bir sinyal işlem tarafından bradyzoite adında bir yavaş büyüyen encysted formu dönüştürmek. Hayvanlar içinde merkezi sinir sistemi ve kas dokusu, bradyzoite kist bulundu ve kronik enfeksiyon aşamasını temsil. Bradyzoites Dönüşüm yüksek pH ya da interferon gamma (IFNγ) yanı sıra orta kullanarak CO 2 açlık doku kültürü simüle edilebilir. Bradyzoites lektin Dolichos tohumları biflorus agglutinin (DBA) bağlayan bir kist duvarının varlığı ile karakterizedir. Floresan ile işaretlenmiş DBA düşük CO 2 ve yüksek pH ortamı maruz kalmış insan sünnet derisi fibroblastlar (paketleme) yetiştirilen parazitler kist görselleştirmek için kullanılmaktadır. Benzer şekilde, parazitler BMMs IFNγ ve lipopolisakkarid (LPS) ile aktive edildikten sonra iliği kaynaklı makrofajlar (BMMs) DBA tarafından algılanamaz bir kist görüntülemek fare kemik ikamet eden. Bu protokol, T. dönüşüm nasıl ikna etmek için ortaya koyacak BMMs düşük CO 2 ve aktivasyon ile yüksek pH besi kullanarak bradyzoites gondii. Konak hücrelerine lamelleri üzerinde kültüre tachyzoites ile enfekte ve ya IFNγ ve LPS (BMMs) eklenmesi ile aktif veya üç gün boyunca yüksek pH büyüme orta (paketleme) maruz olacaktır. Enfeksiyon tamamlanmasından sonra, konak hücreleri, sabit permeabilize, ve bloke olacaktır. Kist duvarları floresan mikroskobu ile rodamin DBA kullanılarak görüntülendi.

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 doku kültürü modelleri taklit edilebilmektedir çevresel strese yanıt olarak bir kist formuna dönüşür. Bu video makrofajlar kemik iliği kaynaklı aktive veya fibroblast hücreleri büyüme orta pH değerini değiştirerek kist oluşumunu incelemek için teknikler gösterilmiştir.

Aktif Kemik İliği türetilen Makrofajlar ve Bradyzoite Koşullar Toxoplasma gondii Kist Duvar Oluşumu

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

toxoplasma-gondii-cyst-wall-formation-activated-bone-marrow-derived

Research

JoVE Journal

Immunology and Infection

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

16.1K Görüntüleme.  University of Wisconsin. Toxoplasma gondii doku kültürü modelleri taklit edilebilmektedir çevresel strese yanıt olarak bir kist formuna dönüşür. Bu video makrofajlar kemik iliği kaynaklı aktive veya fibroblast hücreleri büyüme orta pH değerini değiştirerek kist oluşumunu incelemek için teknikler gösterilmiştir.

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

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with causing food- and water-borne disease outbreaks. The definitive host is the feline species where sexual replication occurs resulting in the development of the highly infectious and environmentally resistant oocyst. Infection occurs via ingestion of tissue cysts from contaminated meat or oocysts from soil or water. Infection is typically asymptomatic in healthy individuals, but results in a life-long latent infection that can reactivate causing toxoplasmic encephalitis and death if the individual becomes immunocompromised. Meat contaminated with T. gondii cysts have been the primary source of infection in Europe and the United States, but recent changes in animal management and husbandry practices and improved food handling and processing procedures have significantly reduced the prevalence of T. gondii cysts in meat1, 2. Nonetheless, seroprevalence in humans remains relatively high suggesting that exposure from oocyst contaminated soil or water is likely. Indeed, waterborne outbreaks of toxoplasmosis have been reported worldwide supporting the theory exposure to the environmental oocyst form poses a significant health risk3-5. To date, research on understanding the prevalence of T. gondii oocysts in the water and environment are limited due to the lack of tools to detect oocysts in the environment 5, 6. This is primarily due to the lack of efficient purification protocols for obtaining large numbers of highly purified T gondii oocysts from infected cats for research purposes. This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation7.

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation

Yüksek Saflaştırılmış Alınması Toxoplasma gondii Süreksiz Sezyum Klorür Gradient ookistleri

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with ...

İmmünoloji ve Enfeksiyon

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways1-5. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca2+, a signal that is also critical for host cell invasion6-8. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress9. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches10-12. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress13. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma biology11,14,15, only recently has genetic mapping of mutations underlying the phenotypes become routine16-18. Furthermore, by generating temperature-sensitive mutants, essential processes can be dissected and the underlying genes directly identified. These mutants behave as wild-type under the permissive temperature (35 &deg;C), but fail to proliferate at the restrictive temperature (40 &deg;C) as a result of the mutation in question. Here we illustrate a new phenotypic screening method to isolate mutants with a temperature-sensitive egress phenotype13. The challenge for egress screens is to separate egressed from non-egressed parasites, which is complicated by fast re-invasion and general stickiness of the parasites to host cells. A previously established egress screen was based on a cumbersome series of biotinylation steps to separate intracellular from extracellular parasites11. This method also did not generate conditional mutants resulting in weak phenotypes. The method described here overcomes the strong attachment of egressing parasites by including a glycan competitor, dextran sulfate (DS), that prevents parasites from sticking to the host cell19. Moreover, extracellular parasites are specifically killed off by pyrrolidine dithiocarbamate (PDTC), which leaves intracellular parasites unharmed20. Therefore, with a new phenotypic screen to specifically isolate parasite mutants with defects in induced egress, the power of genetics can now be fully deployed to unravel the molecular mechanisms underlying host cell egress.

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

Forward genetics is a powerful method to unravel the molecular level of how Toxoplasma egresses from its host cell. Protocols are provided to chemically mutagenize parasites, enrich for mutants with defects in induced egress, and validate the phenotype of cloned mutants.

Isolate A Genetik Ekran Toxoplasma gondii Host-hücre çıkış Mutants

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth ...

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

Kemik İliği Türetilmiş makrofajlar kullanarak Makrofaj Polarizasyon İncelenmesi

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The &Delta;ku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The &Delta;ku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.
Here, we report methods for using type I and type II &Delta;ku80&Delta;hxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in &Delta;ku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Genetic Manipulation in &lt;em&gt;&amp;Delta;ku80&lt;/em&gt; Strains for Functional Genomic Analysis of &lt;em&gt;Toxoplasma gondii&lt;/em&gt;

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

Genetik Manipülasyon<em&gt; Δku80</emFonksiyonel Genomik Analiz&gt; Suşlarının<em&gt; Toxoplasma gondii</em

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Kemik İliği Makrofaj üretim kaynaklı

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

İzolasyon ve Kemirgen Kemik İliği intravenöz enjeksiyon Türetilmiş Monositler

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within virtually any warm blooded vertebrate cell type. During parasite invasion of a host cell, the parasite creates a parasitophorous vacuole (PV) that originates from the host cell membrane independent of phagocytosis within which the parasite replicates. While IFN-dependent-innate and cell mediated immunity is important for eventual control of infection, innate immune cells, including neutrophils, monocytes and dendritic cells, can also serve as vehicles for systemic dissemination of the parasite early in infection. An approach is described that utilizes the host innate immune response, in this case macrophages, in a forward genetic screen to identify parasite mutants with a fitness defect in infected macrophages following activation but normal invasion and replication in na&#239;ve macrophages. Thus, the screen isolates parasite mutants that have a specific defect in their ability to resist the effects of macrophage activation. The paper describes two broad phenotypes of mutant parasites following activation of infected macrophages: parasite stasis versus parasite degradation, often in amorphous vacuoles. The parasite mutants are then analyzed to identify the responsible parasite genes specifically important for resistance to induced mediators of cell autonomous immunity. The paper presents a general approach for the forward genetics screen that, in theory, can be modified to target parasite genes important for resistance to specific antimicrobial mediators. It also describes an approach to evaluate the specific macrophage antimicrobial mediators to which the parasite mutant is susceptible. Activation of infected macrophages can also promote parasite differentiation from the tachyzoite to bradyzoite stage that maintains chronic infection. Therefore, methodology is presented to evaluate the importance of the identified parasite gene to establishment of chronic infection.

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&amp;#947;-Dependent Cell Autonomous Immunity

Forward genetics is a powerful approach to identify genes in intracellular pathogens important for resistance to cell autonomous immunity. The current approach uses innate immune cells, specifically macrophages, to identify novel Toxoplasma gondii genes important for immune evasion.

İleri Genetik tanımak makrofajlar kullanılarak Ekranlar<em&gt; Toxoplasma gondii</em&gt; Genler önemli direnç için IFN-γ-bağımlı hücre otonom bağışıklık

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within ...

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage&#8217;s metabolism and phenotype.

Gerçek zamanlı Ekstrasellüler Flux Analizi Polarize M1 ve M2 Kemik İliği-türevli Makrofagların Metabolik Karakterizasyonu

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

Inflammasome harekete geçirmek ve Pyroptotic hücre ölümü fare kemik iliği türevi makrofajlar içinde tespiti

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and adaptive immune responses. In vitro study of macrophages can be undertaken by ex vivo culture from the peritoneum or through differentiation of myeloid bone marrow progenitor cells to form bone marrow-derived macrophages (BMDMs). A common approach to macrophage differentiation from precursors involves the use of conditioned media from L929 cells (LCM). This media is easy to self-produce but suffers from batch variability, and its constituents are undefined. Similarly, Foetal Bovine Serum (FBS) is used to support growth but contains a vast mixture of undefined molecules that may vary between batches. These methods are not adequate for the study of nitric oxide biology and redox mechanisms as they both contain substantial amounts of small molecules that either interfere with redox mechanisms or supplement levels of cofactors, such as tetrahydrobiopterin (BH4), required for the production of NO from inducible nitric oxide synthase (iNOS). In this report, we present an optimized protocol allowing for control of the NO-redox environment by reducing the levels of exogenous biopterin while maintaining conditions suitable for cell growth and differentiation. Tight control of culture media composition helps ensure experimental reproducibility and facilitates accurate interpretation of results. In this protocol, BMDMs were obtained from a GTP cyclohydrolase (GCH)- deficient mouse model. Culture of BMDMs was performed with media containing either (i) conditioned LCM, or (ii) recombinant M-CSF and GM-CSF to produce minimal artifacts while obtaining BH4 and NO-deficient culture conditions - thus allowing for the reproducible study of NO-redox biology and immunometabolism in vitro.

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology

This protocol has been established to culture tetrahydrobiopterin (BH4)- and inducible nitric oxide synthase (iNOS)-deficient primary murine macrophages to study NO-redox biology. The study focuses on reducing potential contamination of BH4 and other artifacts found in traditional isolation and culture methods which may confound experimental outcomes and interpretation of results.

NO-Redoks Biyolojisi Çalışması için Kemik İliği Kaynaklı Makrofajların İzolasyonu ve In vitro Kültürü

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and ...