이 작품은 위스콘신 - 매디슨 허먼 I. 샤피로 탁월한 연구 원정대 (CMT), NIH 수상 AI072817 (AMP)와 미국 심장 협회 수상 0840059N (LJK)의 대학에 의해 지원되었다.

1. 인간의 포피의 섬유아 세포 (HFF) 코팅 coverslips의 준비 <ol><li> 24 잘 조직 문화 판의 우물의 바닥에 살균 둥근 유리 coverslip를 놓습니다. </li><li> 합류 150cm 두 플라스크에서 HFFs을 수확하려면, 1X PBS로 두 번 술병을 씻어 0.025 % 트립신 - EDTA (에틸렌 다이아 민 테트라 초산) 2.5 ML를 추가합니다. ° C 50~10분에 대한 37 휴대용 술병을 품어. </li><li> 손바닥에 대한 술병의 측면을 감청하는 것은 술병에서 세포를 분리에 도움하실 수 있습니다. 일단 세포가 술병에서 발표했습니다 HFF 중간 150 ML (Dulbecco의 수정된 이글 매체 [DMEM]이 10% FBS와 2 MM L - 글루타민,, 1 % 페니실린 - 스트렙토 마이신)과 그 술병을 씻어 수집 매체를 사용을 추가 세포. </li><li> coverslips 잘 당 세포의 1 ML 하는걸. </li><li> 세포가 37 ° C 5 % CO 2 합류가 될 수 있습니다. </li></ol> 2. BMC 개발 L929 에어컨 매체 (CM)를 준비 <ol><li> L929은 confluency 오랜 기간 후에 요소를 자극 대식 세포 콜로니를 secretes murine fibrosarcoma aneuploid의 셀 라인이다. 이 분비는 세포 배양 1 macrophages에 마우스 골수 세포를 개발하는 데 사용할 L929 세포에서 CM 수 있습니다. </li><li> L929 세포가 합류되면, 그들은 구형 나타나고 술병을 리프팅 시작할 때까지 그들이 추가 7~9일에 대해 품어 보자. 뜨는 및 펠렛 10 분 425 XG든지 분리된 세포를 수집합니다. 이 뜨는은 CM입니다. </li><li> L929 세포 중 하나는 150cm이 플라스크는 BMC 매체 150 ML를 생성 CM 30 ML을 산출. BMC 매체 10% FBS, DMEM에 20 %의 CM, 1 % 페니실린 - 스트렙토 마이신,, 1 % L - 글루타민으로 구성되어 있습니다. </li></ol> 3. 골수 및 BMCs의 세포 배양의 분리 <ol><li> CO 2 질식 및 표면 70 % 에탄올로 소독 한 C57BL / 6 마우스를 희생. </li><li> 다리 근처의 복부 피부를 리프팅과 가위로 절단하여 복막을 폭로. 복막 구멍을 펑쳐링하지 마십시오. 발목 부분의 경골과 대퇴골을 폭로하기 위해 다리 아래로 잘라. 가위를 사용하여 다리의 뼈가 근육 조직을 잘라 버릴거야. </li><li> 대퇴골을 제거하려면 한 번 무릎 아래와 한번 엉덩이 근처 컷. 차가운 1X PBS를 포함하는 세균 배양 접시에있는 뼈를 놓습니다. BMCs 영구적으로 준수하기 때문에 다음 단계를위한 페트리 요리를 대접 조직 문화를 사용하지 마십시오. 추가 근육 조직을 긁어 메스를 사용합니다. 그냥 골수를 노출하기 위해 무릎 위의 뼈를 잘라. </li><li> 단계를 반복 (B) 그리고 (c) 두 번째 대퇴골의 골수를 수집합니다. </li><li> 차가운 1X PBS와 50 ML 원뿔 관에 골수를 이동시키다하는 25 게이지 바늘로 가득한 10cc 주사기를 사용하십시오. 뼈를 새하얀 한 골수이 제거됩니다 나타납니다. </li><li> 모든 골수 집계를 깨고 22 게이지 바늘을 통해 PBS / 골수 혼합물 패스. </li><li> 10 분 425 XG에서 혼합 스핀, 그리고 표면에 뜨는를 제거합니다. </li><li> BMC 매체 8 ML에서 펠렛을 Resuspend. 8 세균 배양 접시에 세포 현탁액 9 ML BMC 매체 1 ML을 추가합니다. </li><li> 37 품어 ° C 5 % CO 2. </li><li> 5 일째, 각 접시 10 ML BMC 매체를 추가합니다. 일 7.2으로 전지가 완전히 개발됩니다. BMCs는 성숙 후 1-2주에 대한 passaged 수 있습니다. </li><li> BMCs을 분할하려면 미디어를 제거하고 각 페트리 접시에 차가운 1X PBS의 5 ML를 추가합니다. 4 품어 ° 세포 리프트 시작까지 30 분 C. 멸균 전송 피펫과 접시에서 BMCs을 씻어. </li><li> 50 ML 원뿔 관 및 펠릿 10 분 425 XG에 수영장에서 BMCs합니다. </li><li> 10 ML BMC 매체와 hemocytometer 의지의 펠렛을 Resuspend. 바닥에 둥근 유리 coverslips로 24 잘 플레이트에 잘마다 씨앗 2x105 BMCs. T.과 감염 전에 BMCs가 밤새 준수합시다 gondii. 초과 BMCs는 나중에 사용하기 위해 배양 접시에 reseeded 수 있습니다. </li></ol> 4. T.과 감염 세포 gondii <ol><li> 2x10 6 T.과 합류 HFFs의 25cm 두 플라스크를 감염 세포 (2-3 일 정도) lyse 시작할 때까지 gondii하고 성장합니다. </li><li> 조직 문화 플라스크에서 감염된 숙주 세포의 monolayer를 제거하는 세포 스크레이퍼를 사용하여 다음 27 게이지 바늘을 통해 dislodged monolayer를 전달하여 호스트 세포에서 기생충을 놓습니다. </li><li> 매체에 기생충의 수를 결정하는 hemocytometer를 사용합니다. </li><li> 프로토콜 1 또는 프로토콜 3 BMCs에서 HFF monolayers의 coverslip 잘 당 10 5 기생충 감염. 기생충, 37에서 3 시간 동안 침략 ° C 5 % CO 2를 보자. </li></ol> 5. T.을 시작 환경 스트레스에 의해 gondii bradyzoite 개발 <ol><li> 중간 산도 증가와 CO 2 기아와 HFFs에 Bradyzoite 개발 <ol class="lalpha"><li> 개발 매체를 준비합니다. 개발 매체 RPM을 포함I 중탄산염없이 1640, 1 % FBS, 1 % 페니실린 - 스트렙토 마이신, 42 MM HEPES. 8.0 산도와 필터 소독. </li><li> 감염된 HFFs에서 DMEM 제거 1X PBS와 린스, 개발 매체 1ml를 추가합니다. </li><li> 주변 공기와 함께 37 삼일 ° C에 대해 숙고하다. </li></ol></li><li> BMCs의 활성화 <ol class="lalpha"><li> BMCs을 활성화하기 위해 매체를 준비합니다. 활성화 매체 100U/ml IFN - γ와 100 NG / ML LPS와 함께 보충 BMC의 매체입니다. 집계를 중단하기 위해 사용하기 전에 2 분 동안 물을 욕조에 LPS를 Sonicate. </li><li> 우물에서 BMC 매체를 제거하고 활성 매체 1 ML로 바꿉니다. </li><li> 37 품어 ° C, 5 % 3 일간 CO 2. </li></ol></li></ol> 6. Immunofluorescence 검출 <ol><li> 1X PBS에 감염된 우물을 세 번 씻어. </li><li> 20 분 3 % 포름 알데히드의 200ul와 monolayer을 수정. </li><li> 정착액을 제거하고 1X PBS로 우물을 씻어. </li><li> 우물에 0.1 M 글리신의 200μl를 추가하고 5 분 앉아 보자. </li><li> 글라 이신를 제거하고 1X PBS로 우물을 씻어. </li><li> 30 분 monolayer를 permeabilize하고 차단하는 3퍼센트 BSA/0.2 % 1X PBS에서 TritonX - 100 250 μl를 추가합니다. </li><li> 차단 솔루션을 제거하고 1X PBS로 우물을 씻어. </li><li> 3퍼센트에 rhodamine Dolichos biflorus agglutinin의 1:250 희석의 50 μl를 추가 BSA/0.2 % 트리톤 1X PBS에서 X - 100. </li><li> 1 시간 동안 실온에서 플랫폼에 쉐이크 판과 장소를 커버 </li><li> 0.2 %로 coverslips에게 3 번 씻어 튼과 각 플랫폼 쉐이크 5 분 1X PBS에서 X - 100. </li><li> 4&#39;6 - diamidino - 2 - phenylindole (DAPI)를 포함하는 VectaShield 장착 매체를 사용하여 마운트 coverslips. </li><li> 스테인드 T. gondii는 100x의 확대에 rhodamine에 대한 필터를 적절한 형광 현미경을 사용하여 시각하실 수 있습니다. </li></ol> 7. 대표 결과 그림 1은 T.의 대표적인 DBA의 염색법을 보여줍니다 산도 응력 하에서 활성화 BMMs과 HFFs에 gondii. 기생충 주변 모두보기 DBA의 얼룩은 낭종 벽 구성 요소의 존재를 나타내는 vacuoles을 포함. 활성화 BMM 이미지는 공포의 표면과 일치 DBA 염색법을 보여줍니다. T.의 단면 gondii는 산도에 HFFs은 스테인드없이 내부 구조와 낭종 벽을 보여줍니다 강조했다. <img src="/files/ftp_upload/2091/2091fig1.jpg" alt="그림 1" /> 그림 1. 스트레스 T.의 DBA 염색법 스트레스 조건, 활성화 BMMs이나 산도에 따라 gondii. 세포내 기생충, HFFs 강조 rhodamine 복합 DBA (적색)와 스테인드되었습니다. 차동 간섭 대비 (DIC)은 낭종의 개요를 보여줍 검은 스케일 바는 2 μm의 같습니다. 동물 실험 동물 실험 위스콘신 애니멀 케어 및 사용위원회의 대학에 의해 명시된 지침과 규정에 따라 수행되었다.

<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>

관심 없음 충돌 선언하지 않습니다.

T.의 bradyzoite 개발의 메커니즘은 완전히 이해되지 않지만, 분자 유전 분석 조직 문화 gondii 단계 변환 bradyzoite의 낭종 형성 2,3,4에 관련된 유전자의 발견되었다. 분석은 또한 일부 bradyzoite 마커가 활성화 macrophages 5,6 성장 등 다른 장기 응력 조건에서 표현되는 관찰되었다. 위의 방법은 T. 성장하는 방법을 설명합니다 gondii와 유도 발전 BMCs과 HFFs 모두에서 DB...

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		<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 gondii는 온혈 동물의 nucleated 세포에 침입할 수있는 의무 세포내 기생충이다. 감염 동안, T. 빠른 복제 양식이 tachyzoite 전화로 gondii는 disseminates. Tachyzoites 잘 묘사되지 않은 신호 처리에 의해 bradyzoite라는 느린 성장 encysted 형식으로 변환합니다. 동물 내에서 bradyzoite의 cysts는 중추 신경계와 근육 조직에서 발견하고 감염의 만성 단계를 대표하고 있습니다. bradyzoites 전환은 높은 산도, 또는 인터페론 감마 (IFNγ)의 추가로 매체를 사용하여 CO 2 기아의 조직 문화에 시뮬레이션 할 수 있습니다. Bradyzoites는 렉틴가 Dolichos biflorus agglutinin (DBA)가 바인딩에 낭종 벽의 존재에 의해 특징입니다. 찬란 분류 DBA는 낮은 CO 2와 높은 산도 매체에 노출되었습니다 인간의 포피의 섬유아 세포 (HFFs) 재배 기생충에있는 낭종 벽을 시각화하는 데 사용됩니다. 마찬가지로, 기생충은 BMMs가 IFNγ와 lipopolysaccharide (LPS)로 활성화한 후 골수 파생 macrophages (BMMs)가 DBA에 의해 감지 낭종 벽을 표시 murine 뼈에 거주. 이 프로토콜은 T.의 전환을 유도하는 방법을 보여줍니다 것입니다 BMMs의 낮은 CO 2 및 활성화와 높은 산도 성장 매체를 사용하여 bradyzoites하는 gondii. 호스트 세포는 coverslips에 대한 교양 tachyzoites와 감염 중 IFNγ와 LPS (BMMs)를 추가로 활성이나 사흘 동안 높은 산도 성장 매체 (HFFs)에 노출됩니다. 감염 완료되면, 호스트 세포는 고정 per​​meabilized 및 차단됩니다. 낭종 벽은 형광 현미경과 rhodamine의 DBA를 사용하여 시각 것입니다.

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 gondii는 조직 문화 모델 했었 수있는 환경 스트레스에 대한 응답으로 낭종 형태로 변환됩니다. 이 동영상은 골수 파생 macrophages 뼈를 활성화하거나 fibroblast 세포 성장 매체 산도를 변경하여 낭종 벽 형성을 검토하기 위해 기술을 보여줍니다.

활성 골수 - 유래 Macrophages과 Bradyzoite Toxoplasma gondii 약관의 낭종의 벽 형성

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 조회수.  University of Wisconsin. Toxoplasma gondii는 조직 문화 모델 했었 수있는 환경 스트레스에 대한 응답으로 낭종 형태로 변환됩니다. 이 동영상은 골수 파생 macrophages 뼈를 활성화하거나 fibroblast 세포 성장 매체 산도를 변경하여 낭종 벽 형성을 검토하기 위해 기술을 보여줍니다.

비디오: 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

고도 정화 구하기 Toxoplasma gondii</em불연속 세슘 클로라이드 그라디언트로> Oocysts

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

연구

면역학 및 감염병학

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.

분리하기위한 유전자 스크린 Toxoplasma gondii 호스트 세포 재현의 돌연변이

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.

골수 유래 대 식세포를 사용하여 대식 세포 분극 조사

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.

유전 조작<em&gt; Δku80</em의 기능 게놈 분석을위한&gt; 균주<em&gt; 톡소 포자충</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.

뼈 대식 세포 생산에게 골수 유래

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).

분리 및 설치류 골수의 정맥 주사 유래 단핵구

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.

앞으로 유전학 식별하는 대 식세포를 사용하여 화면<em&gt; 톡소 포자충</em&gt; 유전자 중요 저항이하는 IFN-γ 의존 세포 자치 내성

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.

실시간 세포 외 유출 분석을 사용하여 편광 M1과 M2 골수 유래 대 식세포의 대사 특성

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 활성화 및 Pyroptotic 세포 죽음 Murine 골 수 유래 세포에서의 검출

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-Redox 생물학의 연구를 위한 골수 유래 대식세포의 분리 및 시험관내 배양

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