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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>bisBenzimide H 33342 trihydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>B2261</td>
			<td>Hoechst 33342. Store a 4mM stock solution at -20 &#176;C in distilled water</td>
		</tr>
		<tr>
			<td>Hoechst 34580</td>
			<td>Sigma-Aldrich</td>
			<td>63493</td>
			<td>Store a 2mM stock solution at -20 &#176;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 6mM stock solution at -20 &#176;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 2mg/ml stock solution at -20 &#176;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 25mg/ml stock solution at -20 &#176;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&#181;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

9.5K 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

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

유동세포계측법로 고정하고 살아있는 세포에서 Calpain 활동을 측정

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

연구

면역학 및 감염병학

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

유동세포계측법에 의해 피해를 입은 척수 조직에서 면역 세포의 양적 평가 : 세포 정제 방법에 대한 소설을 사용

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

사람의 체외에서</em면역 불균형의 초기 국가의 인정을위한 심사 도구를> 억제

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

유동 세포 계측법에 의한 Thymic 긍정적이고 부정적인 선택의 시험

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

유동 세포 계측법에 의해 쥐의 신장에서 대 식세포의 표현형 특성

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

망막 미세 아교 식세포의 기능을 평가<em&gt; 생체</em&gt;은 유동 세포 계측법 기반 분석을 사용하여

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

PM10의 입자 바인딩 베팅 1 절 알레르기의 유세포 분석

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

고립과 Murine 인간과 치경 대 식 세포의 생체 외에서 문화

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

전체 혈액에 의해 인간 Monocyte 하위 집합의 Flow Cytometry 분석

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic &#34;one fluorochrome-one marker approach&#34;, although the latter is still more precise for identifying populations such as NKT cells and &#947;&#948; T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

2 형광 색소에 의해 7 개의 면역 세포 하위 집합의 차별 Cytometry 흐름

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...