In vivo evaluatie van het knaagdier Plasmodium parasitemia en merozoïet Invasion van flowcytometrie
Summary
De malariaparasiet binnenvalt en herhalingen binnen de rode bloedcellen. Nauwkeurige vaststelling van de merozoïet invasie en parasitemia is daarom van cruciaal belang bij de beoordeling van het verloop van de malaria-infectie. Hier beschrijven we een flowcytometrie gebaseerd protocol voor het meten van deze parameters in een muismodel van malaria.
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
Representative Results
Discussion
We hebben een werkwijze beschreven voor het meten van zowel parasitemie en merozoïet invasie van in vivo monsters. In termen van parasitemia meting, biedt deze methode een voordeel ten opzichte van eerdere methoden 10-13 in die HJ-rode bloedcellen kan worden onderscheiden van pRBCs, waardoor het aantal valse positieve gebeurtenissen verminderen. Terwijl HJ-RBC's zijn meestal zeldzaam bij de mens, ook onderzoeken met hoge niveaus bij muizen 15,16 het onderscheiden van deze cellen en pR…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Wij erkennen de financiering van de steun van de National Health en Medical Research Council (verlenen APP605524, 490.037 en 1.047.082), de Australian Research Council (verlenen DP12010061), de National Collaborative Research Infrastructure strategie van Australië en de Onderwijs investeringsfonds van het ministerie van Innovatie, Industrie , wetenschap en onderzoek. PML is een ontvanger van een Australische Postgraduate award.
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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