Summary

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

Published: April 05, 2015
doi:

Summary

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.

Abstract

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.

Introduction

The clinical symptoms associated with malaria occur during the Plasmodium parasite’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.

Protocol

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 12시 12분 시간의 명암주기 제어 온도 (21 ° C에서) 주택의 마우…

Representative Results

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?…

Discussion

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?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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.

Materials

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

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Cite This Article
Lelliott, P. M., McMorran, B. J., Foote, S. J., Burgio, G. In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry. J. Vis. Exp. (98), e52736, doi:10.3791/52736 (2015).

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