In Vivo Valutazione del roditore Plasmodium parassitemia e merozoite Invasion di Citometria a flusso
Summary
Gli invade parassita della malaria e replicati all'interno delle cellule rosse del sangue. La valutazione accurata di invasione merozoite e parassitemia è quindi fondamentale per la valutazione nel corso di infezione malarica. Qui si descrive un protocollo basato citometria a flusso per la misurazione di questi parametri in un modello murino di 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
Abbiamo descritto un metodo per la misura sia parassitemia e merozoite invasione campioni in vivo. In termini di misurazione parassitemia, questo metodo offre un vantaggio rispetto ai metodi precedenti 10-13 in quella HJ-globuli rossi possono essere distinti da pRBCs, riducendo così il numero di eventi positivi falsi. Mentre HJ-globuli rossi sono di solito rare negli esseri umani, alcuni studi riportano livelli elevati nei topi 15,16 la distinzione tra queste cellule e pRBCs importanti pe…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Riconosciamo il finanziamento il sostegno del Consiglio Nazionale Salute e Ricerca Medica (concedere APP605524, 490.037 e 1.047.082), la Australian Research Council (concedere DP12010061), la strategia di National Infrastructure Research Collaborative d'Australia e il fondo di investimento Education del Dipartimento di Innovazione, Industria , Scienza e la ricerca. PML è un destinatario di un premio post-laurea australiano.
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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