Name: in vivo assessment of rodent plasmodium parasitemia and merozoite invasion by flow cytometry
Uploaded: 2015-04-05T14:00:00.000+00:00
Duration: 7 min 16 s
Description: Watch this Scientific Journal Video about In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry at JoVE.com

우리는 (APP605524, 490,037 및 1,047,082을 부여) 국립 보건 의학 연구위원회 (Medical Research Council)의 지원 자금을 인정 호주 연구 협의회 (DP12010061을 부여), 혁신의 부서에서 호주 국립 공동 연구 인프라 전략 및 교육 투자 기금, 산업 과학 연구. PML은 호주 대학원 상을받는 사람입니다.

모든 절차는 맥쿼리 대학의 정책에 따라 수행과 국민 건강과 의학 연구위원회 (NHMRC) 연습 호주 코드를 본했다. 이 연구는 없음 ARA 2012/017 승인하지 맥쿼리 대학의 동물 윤리위원회에서 얻은 계약 윤리에서 수행되었다. 달리 언급하지 않는 한 모든 실험은 SJL / J 마우스에 대해 수행 하였다. 1. 마우스 및 실험 말라리아 감염 <ol><li> 12시 12분 시간의 명암주기 제어 온도 (21 ° C에서) 주택의 마우스. </li><li> 냉동 보관 P.의 나누어지는 해동 5 % 기생 적혈구 (pRBCs) 및 C57BL / 6 기증자 마우스의 복강 공동으로 200 μl를 주입와 chabaudi 다미의 DS. </li><li> 이 프로토콜의 4 - 3 항에 명시된 유동 세포 계측법 사용하여 2 일 - 매 1 기증자 마우스 기생충을 모니터링합니다. 기증자가 5에 도달하면 - 15 %의 기생충을 다음과 같이 심장 구멍으로 혈액을 수집 : <ol><li>대기음 헤파린 용액 (마우스 링거액 300 U가 / ㎖ 헤파린 (MTR) (154 mM의 염화나트륨, 5.6 mM의 KCl을, 1 ㎜의 MgCl 2, 2.2 mM의 CaCl2를, 20 mM의 HEPES 100 ㎕, 10 mM의 글루코오스, 30 U / ㎖ 헤파린, pH가 7.4, 0.22 μm의 필터는 26 G 바늘 1 ML의 주사기로)) 멸균. </li><li> 페달 탈퇴 응답 및 각막 반사 부재에 의해 마취 원하는 깊이를 확인하는, 5 % 이소 플루오 란으로 흡입에 의한 마우스를 마취. </li><li> 갈비뼈를 통해, 단지 팔꿈치 아래에 옆으로 마우스와 수직으로 삽입 바늘을 놓고 마음에. 천천히 주사기의 플런저를 잡아 당겨 0.5까지 바늘을 회전 - 혈액 1ml를 얻을 수있다. </li><li> 마취하에 자궁 전위에 의해 인도적인 안락사를 수행합니다. </li></ol></li><li> 희생 할 때 기증자가 10 % 기생충에 있다면, 혈액이 9 × 10 (5)를 포함 즉, 9 × 10 (6) RBC / μL (의 혈액 검사를 가정 기증자 혈액의 볼륨 당 pRBCs을 계산pRBC / μL). 크렙스에 기생 혈액 희석은 (25 mM의 NaHCO3을, 1.2mm의의 NaH 2 PO 4, 1.2 mM의의 MgCl 2, 250 mM의 CaCl2를, 0.2 % 글루코스, pH가 7.2, 멸균 0.22 μM 필터 126 mM의 염화나트륨, 2.5 mM의 KCl을) 완충 염수 농도는 200 μL 당 1 × 4 pRBCs하고 RBC 라벨 및 수혈 실험에 필요한 마우스의 복강 공동으로 200 μl를 주입 있도록. </li><li> 이 프로토콜의 4 - 3 항에 기술 된 바와 같이 유동 세포 계측법 사용하여 2 일 - 매 1 기생충을 모니터링합니다. </li></ol> 적혈구 수혈 2. 라벨링 <ol><li> 1.3 단계에 설명 된대로 심장 천자에 의해 마우스의 헤파린 혈액을 수집하고 얼음에 배치합니다. 항상 4 ° C에서 혈액을 보관하십시오. 마우스 당 약 200 ㎕의 혈액을 수집하는 주입한다. 필요한 경우, 두 개의 샘플로 혈액을 분할하고 원하는대로 하나의 샘플을 취급합니다. 주 : 처리 SAMP 이러한 방식으로, 침입르가 대조군에 비해 수 미처리 샘플 (회로도도 1 참조). </li><li> 각각의 형광 RBC 라벨의 2 배 솔루션을 준비합니다. <ol><li> 농도가 20 ㎍ / ㎖의 MTR되도록 아토에서 633-N 히드 록시 숙신이 미드 (NHS-아토 633)의 2 ㎎ / ㎖의 스톡 용액을 희석. </li><li> 농도가 250 ㎍ / ml 인 것을되도록 MTR 비오틴 N 히드 록시 (비오틴 NHS)의 25 ㎎ / ㎖의 스톡 용액을 희석. </li></ol></li><li> 두 튜브에 혈액 시료를 분할 한 관 및 다른 튜브 배 비오틴 NHS 동일 부피의 633 배까지 아토-NHS 용액 동량을 추가한다. 즉시 섞는다. 일정 느린 혼합 1 시간 동안 4 ° C에서 혈액을 품어, 또는 대안 적으로, 매 10 샘플을 혼합 - 15 분. </li><li> 다음 MTRC (MTR + 0.5 % 소 혈청 알부민 (BSA), 멸균 필터 0.22 μM)로 표지 된 혈액 3 회 반복한다 : <ol><li> 5 분 750 XG에 MTRC의 최소 2 볼륨, 원심 분리기를 추가 상층 액을 제거하고 resuspenMTRC의 2 개 이상의 볼륨에 펠렛을 거라고. 2 번 이상 반복합니다. </li><li> 5 분 750 XG에 최종 세척, 원심 분리 후 다른 조합 펠렛 혈액을 결합 (즉, 치료 아토 633 / 처리 비오틴, 치료 비오틴 / 아토 (633) 처리). 주입 마우스에 200 μL의 볼륨을 만들기 위해 MTR을 추가합니다. </li></ol></li><li> 다음과 같이 혈관 내 설치류 말라리아에 감염된 마우스 또는 감염되지 않은 컨트롤에 표시된 혈액을 주입한다 : <ol><li> 기생충 schizogony의 피크에서 15 %의 기생충 - 2에서 수혈을 수행합니다. 참고 :이 어둠 사이클을 중간 쯤에 발생합니다 (즉, 12 사이 - 후진 등 사이클 오후 2 - 보통의 빛 사이클 오전 2, 12). </li><li> 5-10 분 동안 가열 램프를 사용하여 따뜻한 생쥐 혈액 흐름을 증가 시키지만 생쥐 과열하지 않도록주의를 기울일. 금지 장치에 마우스를 놓고 혈관 내 200 μL라고 표시된 혈액을 주입 (약 1 - 2 × 10 9 RB꼬리 정맥을 통해 CS). </li></ol></li><li> 제 3 항에 기재된 바와 같이 주입 후 상이한 시점에서 혈액 샘플을 수집. 참고 : 침공 요금은받는 사람 마우스의 기생충에 따라, 주사 후보다 10 분을 정량화 할 수있다. </li></ol> 유동 세포 계측법에 대한 혈액 샘플 및 준비 3. 컬렉션 <ol><li> 다음 실험의 당일에, 염색 용액 샘플 당 플러스 여분의 50 μl를 준비한다 : <ol><li> 디메틸 설폭 사이드에서 -20 ° C (DMSO)에 저장된 6 mM의 JC-1 분취 제상. </li><li> 37 ° C에 따뜻한 50 샘플 당 MTRC의 μL, 플러스 추가. </li><li> 최종 농도가 12 μM이되도록 지속적으로 미리 예열 MTRC를 텍싱 동안, JC-1을 추가합니다. 이는 JC-1 응집체의 형성을 감소시키기 위해 수행된다; 골재 형성 경우,이 염색을 차단하므로하지 원심 분리 관을한다. 참고 : 집계 소량의 결과에 영향을 미치지 않습니다. </li><li> 안티 추가CD45 APC eFluor 780 및 항 CD71를 PerCP eFluor 710 최종 농도 1 ㎍ / ml 인 것을되도록. 표지 된 RBC 실험을 수행하면 최종 농도가 1 ㎍ / ㎖의 수 있도록, 스트렙 타비 딘 PE-Cy7을 추가합니다. </li></ol></li><li> 꼬리 내 혈관의 꼬리 또는 열상의 선단의 절단에 의해 꼬리 출혈을 수행한다. 작은 무게 보트 또는 유리 슬라이드에 꼬리 방울의 피를 놓습니다. 혈액 수집 후, 출혈이 중지되었는지 확인합니다. 염색 액의 50 μL에 꼬리 혈액의 피펫 3 μL는 37 ° C로 예열하고 20 분 동안 37 ° C에서 샘플을 품어. </li><li> (355 nm의 레이저를 사용하는 경우)를 증류수에서 -20 ° C에 보관 4mM의 훽스트 33342 또는 (405 nm의 레이저를 사용하는 경우)를 증류수에서 -20 ° C에 보관 2 mM의 훽스트 34580의 분취 제상. 각각 MTRC의 훽스트 (Hoechst)의 4 μM 또는 2 μM 용액, 샘플 당 500 μl를 준비, 플러스 추가. </li><li> 4 μM 훽스트 (Hoechst)의 500 μl를 추가33342 또는 샘플에 2 μM의 Hoechst 34580 및 실온에서 20 분 동안 배양한다. </li><li> 4 ° C에서 3 분 750 XG, 폐기 뜨는, 원심 분리기 세포는 700 μL MTRC를 추가하고 유동 세포 계측법에 의해 분석한다. </li></ol> 4. 유동 세포 계측법 <ol><li> 355/488/633 nm의 레이저 또는 405/488/633 nm의 레이저 장비를 사용하여 샘​​플을 분석 할 수 있습니다. </li><li> 분석 직전 35 μm의 셀 스트레이너를 통해 필터 샘플. 샘플 및 재사용을 사이에 증류수로 셀 스트레이너를 씻어. </li><li> (- 샘플 당 10,000,000 총 이벤트 일반적으로 1,000,000) 가장 작은 인구가 최소 500 이벤트를 포함하도록 충분한 이벤트를 기록합니다. </li><li> 355 nm의 레이저를 사용 훽스트 33342 익사이트 50분의 460 및 필터를 통해 이벤트를 검출한다. 405 nm의 레이저를 사용 훽스트 34580 익사이트 50분의 460 및 필터를 통해 이벤트를 검출한다. 여기 JC-1, 항 CD71를 PerCP eFluor 710 스트렙 타비 딘 PE-Cy7는 488 nm의 레이저를 사용하여 40분의 530를 통해 이벤트를 탐지, 40분의 692, 및 750LP는 각각의 필터LY. 633 nm의 레이저를 이용하여 아토 (633) 및 항 CD45 APC eFluor 780 익사이트 각각 30분의 670 또는 750LP 필터를 통해 이벤트를 감지합니다. </li><li> 다음과 같이 분석 소프트웨어 세포 계측법 적절한 흐름을 사용하여 분석 및 보정을 수행합니다 <ol><li> 전체 셀을 선택하고 FSC / SSC 특성 (그림 2A)를 기반으로 소음, 먼지, 혈소판을 제외 할 수 있습니다. 어느 트리거 펄스 폭 (도 2b) 또는 높이의 비율 (도 2C)로 FSC 피크 면적을 사용하여 단일 세포를 기반으로 선택한다. </li><li> 성숙한 적혈구를 선택하고를 PerCP eFluor (710)와 APC eFluor 780 채널 (그림 2D)에서 음의 형광을 기반으로 적혈구 전구 세포와 백혈구를 제외합니다. </li><li> 표지 된 RBC 실험을 수행하는 경우, PE-Cy7 및 아토 633 형광을 기반으로 표시된 적혈구의 각각의 인구를 선택하고이 별도로 (그림 3A)를 분석 할 수 있습니다. </li><li> (두 JC-1과 훽스트 (Hoechst)에 대한 긍정적 인 형광 기반으로 pRBCs를 선택 <str옹&gt;도 2e-H). </li></ol></li></ol> 5. 계산 및 통계 <ol><li> 적혈구의 전체 숫자로 pRBCs의 수를 분할함으로써 기생충을 계산 (즉, 게이트 G4의 이벤트로 나눈 게이트 Q2의 이벤트의 수). 두 표지 개체군 침입 검정 기생충을 수행 할 때 (즉, 집단에서 표지 된 RBC의 숫자로 표시된 pRBCs의 개수를 나눔으로써 계산 될 수있다, 즉, 게이트들 L1 및 Q2 모두에있는 이벤트의 수, 이벤트로 나눈 게이트 L1에서). 참고 : 레이블이 기생충은 크게 내인성 기생충 및 순환 merozoites의 수 등의 요인에 따라 달라집니다. 이 때문에, 각 개별 마우스에 표시된 제어 인구의 기생충에 의해 분할 처리 표지 인구 기생충로서 계산된다 기생충 율을보고하는 것이 도움이된다. 가능한 염료 효과를 해결하려면, 보고서 결과두 염료 조합에서 평균 기생충 비율이야. </li><li> 가상의 수단으로 1 일 표본 t의 -test를 사용 비율의 통계적 유의성을 확인합니다. </li></ol>

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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+flow+cytometry-based+assay+for+measuring+invasion+of+red+blood+cells+by+Plasmodium+falciparum.">A flow cytometry-based assay for measuring invasion of red blood cells by Plasmodium falciparum.</a> Am J Hematol. 85 (4), 234-237 (2010).</li><li>Clark, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RBC+barcoding+allows+for+the+study+of+erythrocyte+population+dynamics+and+P.+falciparum+merozoite+invasion.">RBC barcoding allows for the study of erythrocyte population dynamics and P. falciparum merozoite invasion.</a> PLoS One. 9 (7), e101041 (2014).</li><li>Malleret, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+robust+tri-color+flow+cytometry+assay+for+monitoring+malaria+parasite+development.">A rapid and robust tri-color flow cytometry assay for monitoring malaria parasite development.</a> Sci Rep. 1 (118), (2011).</li><li>Jimenez-Diaz, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurement+of+Plasmodium-infected+erythrocytes+in+murine+models+of+malaria+by+flow+cytometry+using+bidimensional+assessment+of+SYTO-16+fluorescence.">Quantitative measurement of Plasmodium-infected erythrocytes in murine models of malaria by flow cytometry using bidimensional assessment of SYTO-16 fluorescence.</a> Cytometry A. 75 (3), 225-235 (2009).</li><li>Jimenez-Diaz, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+detection+specificity+of+Plasmodium-infected+murine+erythrocytes+by+flow+cytometry+using+autofluorescence+and+YOYO-1.">Improvement of detection specificity of Plasmodium-infected murine erythrocytes by flow cytometry using autofluorescence and YOYO-1.</a> Cytometry A. 67 (1), 27-36 (2005).</li><li>Jun, G., Lee, J. S., Jung, Y. J., Park, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+determination+of+Plasmodium+parasitemia+by+flow+cytometry+and+microscopy.">Quantitative determination of Plasmodium parasitemia by flow cytometry and microscopy.</a> J Korean Med Sci. 27 (10), 1137-1142 (2012).</li><li>Lelliott, P. M., Lampkin, S., McMorran, B. J., Foote, S. J., Burgio, 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+flow+cytometric+assay+to+quantify+invasion+of+red+blood+cells+by+rodent+Plasmodium+parasites+in+vivo.">A flow cytometric assay to quantify invasion of red blood cells by rodent Plasmodium parasites in vivo.</a> Malar J. 13 (1), 100 (2014).</li><li>Morohashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+abnormalities+in+the+spleen+of+an+mFtz-F1+gene-disrupted+mouse.">Structural and functional abnormalities in the spleen of an mFtz-F1 gene-disrupted mouse.</a> Blood. 93 (5), 1586-1594 (1999).</li><li>Shet, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+and+functional+platelet+abnormalities+in+Berkeley+sickle+cell+mice.">Morphological and functional platelet abnormalities in Berkeley sickle cell mice.</a> Blood Cells Mol Dis. 41 (1), 109-118 (2008).</li><li>Duraisingh, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+variation+of+Plasmodium+falciparum+merozoite+proteins+directs+receptor+targeting+for+invasion+of+human+erythrocytes.">Phenotypic variation of Plasmodium falciparum merozoite proteins directs receptor targeting for invasion of human erythrocytes.</a> EMBO J. 22 (5), 1047-1057 (2003).</li><li>Okoyeh, J. N., Pillai, C. R., Chitnis, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+falciparum+field+isolates+commonly+use+erythrocyte+invasion+pathways+that+are+independent+of+sialic+acid+residues+of+glycophorin+A.">Plasmodium falciparum field isolates commonly use erythrocyte invasion pathways that are independent of sialic acid residues of glycophorin A.</a> Infect Immun. 67 (11), 5784-5791 (1999).</li><li>Dolan, S. A., Miller, L. H., Wellems, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+switching+mechanism+in+the+invasion+of+erythrocytes+by+Plasmodium+falciparum.">Evidence for a switching mechanism in the invasion of erythrocytes by Plasmodium falciparum.</a> J Clin Invest. 86 (2), 618-624 (1990).</li></ol>

저자가 공개하는 게 없다.

우리는 생체 시료의 양 및 기생충 merozoite 침윤 측정하는 방법을 설명 하였다. 기생충 측정의 관점에서,이 방법은 이에 위양성 이벤트의 수를 감소 pRBCs 구별 할 수있는 HJ-적혈구 10-13에서 이전의 방법보다 이점을 제공한다. HJ-적혈구는 인간의 보통 드문 반면, 일부 연구는 설치류 기생충의 정확한 측정을 위해 중요한 이러한 세포와 pRBCs의 구분을 마우스 15, 16의 높은 수준을?...

The clinical symptoms associated with malaria occur during the Plasmodium parasite&#8217;s asexual replicative cycle within red blood cells (RBCs). Merozoites, released during the liver stage of infection, quickly attach to and invade RBCs. After gaining entry into the cell, the parasite grows and matures, eventually undergoing schizogony, splitting open the cell, and releasing a cluster of newly formed merozoites which go on to repeat this cycle. As such, an assessment of malaria infection often involves monitoring both parasitemia, which is the percentage of RBCs appropriated by one or more parasites, and the rate of merozoite invasion into uninfected RBCs. 
Flow cytometry is a powerful tool which can be used to record the properties of vast numbers of cells in a short period of time. This technique has clear applicability for the measurement of malaria parasitemia and invasion, and offers several advantages over traditional microscopy techniques. These include the accurate measurement of very low parasitemia, which would be prohibitively time consuming by microscopy, the unbiased nature of the measurement, and the ability to measure multiple cell parameters simultaneously. Flow cytometry is widely used to determine both parasitemia and merozoite invasion in in vitro culture1-9, however, techniques for measuring these parameters in vivo are less well developed, and can be complicated by the presence of additional cell types which interfere with analysis. No assays have been described for measurement of in vivo invasion, and while some assays exist for the analysis of in vivo parasitemia, these lack the ability to distinguish between parasitized RBCs (pRBCs) and RBCs containing Howell-Jolly bodies (HJ-RBCs)10-13. The later issue is particularly important as in mice HJ-RBCs may account for up to 0.9% of mature RBCs14-16, thereby preventing the accurate measurement of low parasitemia. 
We have previously demonstrated an approach for the measurement of parasitemia and merozoite invasion in a rodent model of malaria infection14. Here, we provide a more detailed protocol and accompanying video. This approach builds on previous methodologies and allows for the accurate identification of parasitized RBCs, as distinct from leukocytes, RBC progenitors, and HJ-RBCs. Additionally, this assay allows the simultaneous measurement of merozoite invasion into two labeled RBC populations, a treated, or target, population, and a control population, thereby providing a robust platform for the assessment of invasion into different cell types.

<table><tbody><tr><td>bisBenzimide H 33342 trihydrochloride</td><td>Sigma-Aldrich</td><td>B2261</td><td>Hoechst 33342. Store a 4 mM stock solution at -20 &amp;deg;C in distilled water</td></tr><tr><td>Hoechst 34580</td><td>Sigma-Aldrich</td><td>63493</td><td>Store a 2 mM stock solution at -20 &amp;deg;C in distilled water</td></tr><tr><td>JC-1 Dye</td><td>Life Technologies</td><td>T-3168</td><td>Store small aliquots of 6 mM stock solution at -20 &amp;deg;C in DMSO</td></tr><tr><td>Anti-Mouse CD45 APC-eFluor 780</td><td>eBioscience</td><td>47-0451-80</td><td>Clone 30-F11</td></tr><tr><td>Anti-Mouse CD71 PerCP-eFluor 710</td><td>eBioscience</td><td>46-0711-80</td><td>Clone R17217</td></tr><tr><td>Atto 633 NHS ester</td><td>Sigma-Aldrich</td><td>1464</td><td>Atto 633-NHS. Store a 2 mg/ml stock solution at -20 &amp;deg;C in DMF</td></tr><tr><td>EZ-Link Sulfo-NHS-LC-Biotin</td><td>Thermo Fisher Scientific</td><td>21335</td><td>Biotin-NHS. Store a 25 mg/ml stock solution at -20 &amp;deg;C in DMF</td></tr><tr><td>Streptavidin PE-Cyanine7</td><td>eBioscience</td><td>25-4317-82</td><td>Streptavidin PE-Cy7</td></tr><tr><td>Heparin</td><td>Sigma-Aldrich</td><td>H478</td><td></td></tr><tr><td>35 &amp;micro;M filter cap tubes</td><td>Becton Dickinson</td><td>352235</td><td></td></tr><tr><td>Flow cytometer: BD LSRFortessa</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>Flow cytometer: BD FACSAria II</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>Flow cytometer: BD Influx</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>Flow cytometer: CyAn ADP Analyzer</td><td>Beckman Coulter</td><td></td><td></td></tr></tbody></table>

in vivo assessment of rodent plasmodium parasitemia and merozoite invasion by flow cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

기생충의 측정. 기생충의 측정을 위해, 혈액 세포를 먼저 선택되어야하고, 소음, 파편 및 혈소판 FSC / SSC 속성 (도 2A)에 기초하여, 배제. 사용 된 세포 계측기에 따라, 단일 세포를 면적비 (도 2c)에 대한 하나의 트리거 펄스 폭 (도 2B), 또는 FSC 피크 높이에 기초하여 선택되어야한다. 이벤트가 백혈구로 구성되어야 남은, APC eFluor 780에 대한 긍?...

Watch this Scientific Journal Video about In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry at JoVE.com

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

적혈구 내에서 말라리아 기생충 침입하여 및 복제합니다. merozoite 침공과 기생충의 정확한 평가는 말라리아 감염의 과정을 평가에 따라서 매우 중요합니다. 여기에서 우리는 말라리아의 마우스 모델에서 이러한 매개 변수의 측정을위한 기반 프로토콜 유동 세포 계측법을 설명합니다.

유동 세포 계측법에 의해 쥐 말라리아 기생충의 생체 내 평가와 Merozoite 침공

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

in-vivo-assessment-rodent-plasmodium-parasitemia-merozoite-invasion

Research

JoVE Journal

Immunology and Infection

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

9.9K 조회수.  Macquarie University.  적혈구 내에서 말라리아 기생충 침입하여 및 복제합니다. merozoite 침공과 기생충의 정확한 평가는 말라리아 감염의 과정을 평가에 따라서 매우 중요합니다. 여기에서 우리는 말라리아의 마우스 모델에서 이러한 매개 변수의 측정을위한 기반 프로토콜 유동 세포 계측법을 설명합니다. 

비디오: In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.

Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

쥐 말라리아 기생충의 유전자 크로스의 생산을위한 프로토콜

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

연구

면역학 및 감염병학

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

휴일 두개골 윈도우를 사용하여 마우스 뇌 엑기스의 Intravital 현미경

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

신경과학

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

비장의 Intravital 현미경 : 기생충 이동성 및 혈류의 정량 분석

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

에서 뇌 - 분리 된 백혈구의 분리 및 분석<em&gt; Plasmodium berghei</em&gt; ANKA에 감염된 마우스

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

높은 수익률 정화<em&gt; 변형체 falciparum의</emOpsonizing 항체 분석 실험에 사용하기 위해&gt; Merozoites

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

<em&gt; 체외</em&gt; HIV 공동 감염의 맥락에서 말라리아에 면역 반응을 측정하기위한 모델

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. However, effective malaria parasite targets for vaccine and chemotherapy development are still limited. This is largely due to the unavailability of relevant and practical in vivo infection models for human Plasmodium species, most notably for P.&#160;falciparum and P. vivax. Therefore, rodent malaria species have been extensively used as practical alternative in vivo models for malaria vaccine, drug targeting, immune response, and functional characterization studies of conserved Plasmodiumspp. genes. Indeed, rodent malaria models have proven to be invaluable, especially for exploring mosquito transmission and liver stage biology, and were indispensable for immunological studies. However, there are discrepancies in the methods used to evaluate the phenotypes of transgenic and wild-type asexual and sexual blood-stage parasites.&#160;Examples of these discrepancies are the choice of an intravenous vs. intraperitoneal infection of rodents with blood-stage parasites and the evaluation of male gamete exflagellation. Herein, we detail standardized experimental methods to evaluate the phenotypes of asexual and sexual blood stages in transgenic parasites expressing reporter-gene or wild-type rodent malaria parasite species. We also detail the methods to evaluate the phenotypes of malaria parasite mosquito stages (gametes, ookinetes, oocysts, and sporozoites) inside Anopheles mosquito vectors. These methods are detailed and simplified here for the lethal and non-lethal strains of P. berghei and P. yoelii but can also be applied with some adjustments to P. chabaudi and P. vinckei rodent malaria species.

Due to the striking similarities of the life cycle and biology of rodent malaria parasites to human malaria parasites, rodent malaria models have become indispensable for malaria research. Herein, we standardized some of the most important techniques used in the phenotypic analysis of wild-type and transgenic rodent malaria species.

설치류 말라리아 기생충 무성 및 성적 혈액 단계 및 모기 단계의 현상형 분석

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

자기 공명 영상을 이용한 실험용 뇌 말라리아 모델에서의 부종 발달 및 미세 혈관 병리학 적 추적

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

의학

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

유동 세포측정 기반 분석체에 의한 플라스모듐 팔시파룸 기생충에 자연적으로 획득된 식세포증-유도 항체 측정

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective immunity to malaria is still poorly understood, mainly due to methodological limitations of long-term Plasmodium culture, especially for Plasmodium vivax. Most studies have focused on adaptive immunity protection against malaria by antibodies, which play a key role in controlling malaria. However, the sterile protection induced by attenuated Plasmodium sporozoites vaccines is related to cellular response, mainly to cytotoxic T lymphocytes, such as CD8+ and gamma delta T cells (&#947;&#948; T). Hence, new methodologies must be developed to better comprehend the functions of the cellular immune response and thus support future therapy and vaccine development. To find a new strategy to analyze this cell-mediated immunity to Plasmodium blood-stage infection, our group established an in vitro assay that measures infected red blood cell (iRBC) killing by cytotoxic lymphocytes. This assay can be used to study cellular immune response mechanisms against different Plasmodium spp. in the blood stage. Innate and adaptative cytotoxic immune cells can directly eliminate iRBCs and the intracellular parasite in an effector:target mechanism. Target iRBCs are labeled to evaluate cell viability, and cocultured with effector cells (CD8+ T, &#947;&#948; T, NK cells, etc.). The lysis percentage is calculated based on tested conditions, compared to a spontaneous lysis control in a flow cytometry-based assay. Ultimately, this killing assay methodology is a major advance in understanding cell-mediated immunity to blood-stage malaria, helping uncover new potential therapeutic targets and accelerate the development of malaria vaccines.

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Here we describe a new method to help elucidate the mechanisms of cellular immunity to Plasmodium during the blood stage of infection. This is an in vitro assay that measures infected red blood cell killing by cytotoxic lymphocytes.&#160;

체외 세포독성 림프구에 의한 변형체 감염 적혈구 사멸 분석

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective ...

유동 세포 계측법에 의해 쥐 말라리아 기생충의 생체 내 평가와 Merozoite 침공

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