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

Summary

The malaria parasite invades and replicates within red blood cells. The accurate assessment of merozoite invasion and parasitemia is therefore crucial in assessing the course of malaria infection. Here we describe a flow cytometry based protocol for the measurement of these parameters in a mouse model of 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.....

Protocol

All procedures were conducted in accordance with the policies of Macquarie University and conformed to the National Health and Medical Research Council (NHMRC) Australian code of practice. The work was performed under the agreement Ethics No ARA 2012/017 approved and obtained from the Animal Ethics Committee at Macquarie University. All experiments were performed on SJL/J mice unless otherwise stated.

1. Mice and Experimental Malaria Infection

1. House mice under controlled temperature (21 °C) with a 12:12 hr light-dark cycle.
2. Thaw an aliquot of cryopreserved P. chabaudi adami DS with 5% parasitized RBCs (pRBCs) and inject 200 &....

Results

Measurement of parasitemia.

For the measurement of parasitemia, blood cells should first be selected, and noise, debris and platelets excluded, based on FSC/SSC properties (Figure 2A). Depending on the cytometer used, single cells should then be selected based on either trigger pulse width (Figure 2B), or FSC peak height to area ratio (Figure 2C). Remaining events should consist of leukocytes, stained positive for APC eFluor 780, RBC progenitors .......

Discussion

We have described a method for the measurement of both parasitemia and merozoite invasion of in vivo samples. In terms of parasitemia measurement, this method offers an advantage over previous methods^10-13 in that HJ-RBCs can be distinguished from pRBCs, thereby reducing the number of false positive events. While HJ-RBCs are usually rare in humans, some studies report high levels in mice^15,16 making the distinction between these cells and pRBCs important for the accurate measurement of rode.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
bisBenzimide H 33342 trihydrochloride	Sigma-Aldrich	B2261	Hoechst 33342. Store a 4 mM stock solution at -20 °C in distilled water
Hoechst 34580	Sigma-Aldrich	63493	Store a 2 mM stock solution at -20 °C in distilled water
JC-1 Dye	Life Technologies	T-3168	Store small aliquots of 6 mM 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 2 mg/ml stock solution at -20 °C in DMF
EZ-Link Sulfo-NHS-LC-Biotin	Thermo Fisher Scientific	21335	Biotin-NHS. Store a 25 mg/ml stock solution at -20 °C in DMF
Streptavidin PE-Cyanine7	eBioscience	25-4317-82	Streptavidin PE-Cy7
Heparin	Sigma-Aldrich	H478
35 µM 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