We acknowledge funding support from the National Health and Medical Research Council (grant APP605524, 490037 and 1047082), the Australian Research Council (grant DP12010061), the National Collaborative Research Infrastructure Strategy of Australia and the Education investment fund from the Department of Innovation, Industry, Science and Research. PML is a recipient of an Australian Postgraduate award.

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
<ol>
	<li>House mice under controlled temperature (21 &#176;C) with a 12:12 hr light-dark cycle.</li>
	<li>Thaw an aliquot of cryopreserved P. chabaudi adami DS with 5% parasitized RBCs (pRBCs) and inject 200 &#181;l into the intraperitoneal cavity of C57BL/6 donor mouse.</li>
	<li>Monitor donor mouse&#160;parasitemia every 1 - 2 days using flow cytometry as described in Section 3 - 4 of this protocol. Once donors reaches&#160;5 - 15% parasitemia, collect blood by cardiac puncture as follows:
	<ol>
		<li>Aspirate 100 &#956;l of heparin solution (300 U/ml heparin in mouse Ringer solution (MTR) (154 mM NaCl, 5.6 mM KCl, 1 mM MgCl2, 2.2 mM CaCl2, 20 mM HEPES, 10 mM glucose, 30 U/ml heparin, pH 7.4, 0.22 &#956;M filter sterilized)) into a 1 ml syringe with a 26 G needle.</li>
		<li>Anesthetize the mouse by inhalation with 5% isofluorane, confirming the desired depth of anesthesia by absence of pedal withdrawal response and corneal reflexes.</li>
		<li>Place mouse on its side and perpendicularly insert needle just below the elbow, through the ribs, and into the heart. Pull out syringe plunger slowly and rotate needle until 0.5 - 1 ml of blood is obtained.</li>
		<li>Perform humane euthanasia by cervical dislocation under anesthesia.&#160;</li>
	</ol>
	</li>
	<li>Calculate the pRBCs per volume of donor blood assuming a blood count of 9 x 106 RBC/&#181;l (i.e., if the donor was at 10% parasitemia when sacrificed, blood will contain 9 x 105 pRBC/&#181;l). Dilute parasitized blood in Krebs buffered saline (126 mM NaCl, 2.5 mM KCl, 25 mM NaHCO3, 1.2 mM NaH2PO4, 1.2 mM MgCl2, 2.5 mM CaCl2, 0.2% glucose, pH 7.2, 0.22 &#956;M filter sterilized) so that the concentration is 1 x 104 pRBCs per 200 &#181;l and inject 200 &#181;l into the intraperitoneal cavity of mice required for the RBC labeling and transfusion experiment.</li>
	<li>Monitor parasitemia every 1 - 2 days using flow cytometry as described in Sections 3 - 4 of this protocol.</li>
</ol>
2. Labeling of RBCs and Transfusion
<ol>
	<li>Collect heparinized blood from mice by cardiac puncture as described in step 1.3, and place on ice. Keep blood at 4 &#176;C at all times. Collect approximately 200 &#181;l blood per mouse to be injected. If necessary, split blood into two samples and treat one sample as desired. 
	NOTE: In this way, invasion of the treated sample can be compared to a control, untreated sample (see Figure 1 for schematic).</li>
	<li>Prepare a 2x solution of each fluorescent RBC label.
	<ol>
		<li>Dilute a 2 mg/ml stock solution of Atto 633-N-Hydroxysuccinimide (Atto 633-NHS) in MTR so that the concentration is 20 &#956;g/ml.</li>
		<li>Dilute a 25 mg/ml stock solution of Biotin-N-Hydroxysuccinimide (Biotin-NHS) in MTR so that the concentration is 250 &#956;g/ml.</li>
	</ol>
	</li>
	<li>Split blood samples into two tubes and add an equal volume of 2x Atto 633-NHS solution to one tube, and an equal volume of the 2x Biotin-NHS to the other tube. Mix immediately. Incubate blood at 4 &#176;C for 1 hr with constant slow mixing, or alternatively, mix sample every 10 - 15 min.</li>
	<li>Wash labeled blood 3 times with MTRC (MTR + 0.5% bovine serum albumin (BSA), 0.22 &#956;M filter sterilized) as follows:
	<ol>
		<li>Add at least 2 volumes of MTRC, centrifuge at 750 x g for 5 min, remove the supernatant, and resuspend the pellet in at least 2 volumes of MTRC. Repeat 2 more times.</li>
		<li>After the final wash, centrifuge at 750 x g for 5 min and combine the pelleted blood in different combinations (i.e., untreated Atto 633/treated Biotin, untreated Biotin/treated Atto 633). Add MTR to make up to a volume of 200 &#181;l per mouse to be injected.</li>
	</ol>
	</li>
	<li>Inject labeled blood intravascularly into rodent malaria infected mice or uninfected controls as follows:
	<ol>
		<li>Perform transfusion at 2 - 15% parasitemia at the peak of parasite schizogony. 
		NOTE: this occurs approximately halfway through the dark cycle (i.e., between 12 - 2 am on a normal light cycle, or 12 - 2 pm on a reverse light cycle).</li>
		<li>Warm mice using a heat lamp for 5-10 mins to increase blood flow, but take precautions not to overheat the mice.&#160;&#160;Place mice in a restraining device and intravascularly inject 200 &#956;l labeled blood (approximately 1 - 2 x 109 RBCs) via the tail vein.</li>
	</ol>
	</li>
	<li>Collect blood samples at different time points after injection as described in Section 3. 
	NOTE: invasion rates can be quantified less than 10 min after injection, depending on parasitemia of the recipient mouse.</li>
</ol>
3. Collection&#160;of Blood Samples and Preparation for Flow Cytometry
<ol>
	<li>Prepare 50 &#956;l of staining solution per sample, plus extra, on the day of the experiment as follows:
	<ol>
		<li>Defrost an aliquot of 6 mM JC-1 stored at -20 &#176;C in dimethyl sulfoxide (DMSO).</li>
		<li>Warm 50 &#956;l of MTRC per sample, plus extra, to 37 &#176;C.</li>
		<li>Whilst continuously vortexing the pre-warmed MTRC, add JC-1 so that the final concentration is 12 &#956;M. This is done to reduce the formation of JC-1 aggregates; if aggregates do form, do not centrifuge tube as this will prevent staining. 
		NOTE: A small amount of aggregation will not affect results.</li>
		<li>Add anti-CD45 APC eFluor 780 and anti-CD71 PerCP eFluor 710 so that the final concentration is 1 &#956;g/ml. If performing the labeled RBC experiment, add streptavidin PE-Cy7 so that the final concentration is 1 &#956;g/ml.</li>
	</ol>
	</li>
	<li>Perform tail bleeding by amputation of the tip of the tail or laceration of the blood vessel within the tail. Place a drop of tail blood onto a small weigh boat or glass slide. After blood collection, ensure that the bleeding has stopped.&#160;&#160;Pipette 3 &#956;l of tail blood into 50 &#956;l of staining solution pre-warmed to 37 &#176;C and incubate samples at 37 &#176;C for 20 min.</li>
	<li>Defrost an aliquot of 4 mM Hoechst 33342 stored at -20 &#176;C in distilled water (if using a 355 nm laser) or 2 mM Hoechst 34580 stored at -20 &#176;C in distilled water (if using a 405 nm laser). Prepare 500 &#956;l per sample, plus extra, of a 4 &#956;M or 2 &#956;M solution of Hoechst in MTRC, respectively.</li>
	<li>Add 500 &#956;l of 4 &#956;M Hoechst 33342 or 2 &#956;M Hoechst 34580 to samples and incubate at RT for 20 min.</li>
	<li>Centrifuge cells at 750 x g for 3 min at 4 &#176;C, discard supernatant, add 700 &#956;l MTRC and analyze by flow cytometry.</li>
</ol>
4. Flow Cytometry
<ol>
	<li>Analyze samples using a 355/488/633 nm laser or 405/488/633 nm laser instrument.</li>
	<li>Filter samples through a 35 &#956;M cell strainer immediately before analysis. Rinse cell strainer with distilled water between samples and re-use.</li>
	<li>Record enough events so that the smallest population contains at least 500 events (usually 1,000,000 - 10,000,000 total events per sample).</li>
	<li>Excite Hoechst 33342 using a 355 nm laser and detect events through a 460/50 filter. Excite Hoechst 34580 using a 405 nm laser and detect events through a 460/50 filter. Excite JC-1, anti-CD71 PerCP eFluor 710 and Streptavidin PE-Cy7 using a 488 nm laser and detect events through a 530/40, 692/40, and 750LP filter respectively. Excite Atto 633 and anti-CD45 APC eFluor 780 using a 633 nm laser and detect events through a 670/30 or 750LP filter respectively.</li>
	<li>Perform analysis and compensation using appropriate flow cytometry analysis software as follows:
	<ol>
		<li>Select whole cells and exclude noise, debris, and platelets based on FSC/SSC properties (Figure 2A). Select single cells based on either trigger pulse width (Figure 2B) or by using the FSC peak area to height ratio (Figure 2C).</li>
		<li>Select mature RBCs, and exclude RBC progenitors and leukocytes, based on negative fluorescence in the PerCP eFluor 710 and APC eFluor 780 channels (Figure 2D).</li>
		<li>If performing the labeled RBC experiment, select each population of labeled RBCs based on PE-Cy7 and Atto 633 fluorescence and analyze these separately (Figure 3A).</li>
		<li>Select pRBCs based on positive fluorescence for both JC-1 and Hoechst (Figure 2E-H).</li>
	</ol>
	</li>
</ol>
5. Calculations and Statistics
<ol>
	<li>Calculate parasitemia by dividing the number of pRBCs by the total number of RBCs (i.e., number of events in gate Q2 divided by number of events in gate G4). When conducting the invasion assay parasitemia of the two labeled populations can be calculated by dividing the number of labeled pRBCs by the number of labeled RBCs in that population (i.e., number of events which are in both gates L1 and Q2, divided by number of events in gate L1). 
	NOTE: Labeled parasitemia varies considerably depending on factors such as endogenous parasitemia and number of circulating merozoites. For this reason, it is helpful to report the parasitemia ratio, which is calculated as the parasitemia of the treated labeled population divided by the parasitemia of the control labeled population in each individual mouse. To correct for possible dye effects, report results as an average parasitemia ratio across the two dye combinations.</li>
	<li>Determine statistical significance of ratios using the one sample t-test with 1 as the hypothetical mean.</li>
</ol>

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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. 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The authors have nothing to disclose.

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 methods10-13 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 mice15,16 making the distinction between these cells and pRBCs important for the accurate measurement of rode...

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><tbody><tr><td>bisBenzimide H 33342 trihydrochloride</td><td>Sigma-Aldrich</td><td>B2261</td><td>Hoechst 33342. Store a 4 mM stock solution at -20 &amp;deg;C in distilled water</td></tr><tr><td>Hoechst 34580</td><td>Sigma-Aldrich</td><td>63493</td><td>Store a 2 mM stock solution at -20 &amp;deg;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 6 mM stock solution at -20 &amp;deg;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 2 mg/ml stock solution at -20 &amp;deg;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 25 mg/ml stock solution at -20 &amp;deg;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 &amp;micro;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.

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 ...

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

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.

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 ...

in-vivo-assessment-rodent-plasmodium-parasitemia-merozoite-invasion

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Immunology and Infection

9.9K Views.  Macquarie University. 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.

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

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.

Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

Neuroscience

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. However, effective malaria parasite targets for vaccine and chemotherapy development are still limited. This is largely due to the unavailability of relevant and practical in vivo infection models for human Plasmodium species, most notably for P.&#160;falciparum and P. vivax. Therefore, rodent malaria species have been extensively used as practical alternative in vivo models for malaria vaccine, drug targeting, immune response, and functional characterization studies of conserved Plasmodiumspp. genes. Indeed, rodent malaria models have proven to be invaluable, especially for exploring mosquito transmission and liver stage biology, and were indispensable for immunological studies. However, there are discrepancies in the methods used to evaluate the phenotypes of transgenic and wild-type asexual and sexual blood-stage parasites.&#160;Examples of these discrepancies are the choice of an intravenous vs. intraperitoneal infection of rodents with blood-stage parasites and the evaluation of male gamete exflagellation. Herein, we detail standardized experimental methods to evaluate the phenotypes of asexual and sexual blood stages in transgenic parasites expressing reporter-gene or wild-type rodent malaria parasite species. We also detail the methods to evaluate the phenotypes of malaria parasite mosquito stages (gametes, ookinetes, oocysts, and sporozoites) inside Anopheles mosquito vectors. These methods are detailed and simplified here for the lethal and non-lethal strains of P. berghei and P. yoelii but can also be applied with some adjustments to P. chabaudi and P. vinckei rodent malaria species.

Due to the striking similarities of the life cycle and biology of rodent malaria parasites to human malaria parasites, rodent malaria models have become indispensable for malaria research. Herein, we standardized some of the most important techniques used in the phenotypic analysis of wild-type and transgenic rodent malaria species.

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

Medicine

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective immunity to malaria is still poorly understood, mainly due to methodological limitations of long-term Plasmodium culture, especially for Plasmodium vivax. Most studies have focused on adaptive immunity protection against malaria by antibodies, which play a key role in controlling malaria. However, the sterile protection induced by attenuated Plasmodium sporozoites vaccines is related to cellular response, mainly to cytotoxic T lymphocytes, such as CD8+ and gamma delta T cells (&#947;&#948; T). Hence, new methodologies must be developed to better comprehend the functions of the cellular immune response and thus support future therapy and vaccine development. To find a new strategy to analyze this cell-mediated immunity to Plasmodium blood-stage infection, our group established an in vitro assay that measures infected red blood cell (iRBC) killing by cytotoxic lymphocytes. This assay can be used to study cellular immune response mechanisms against different Plasmodium spp. in the blood stage. Innate and adaptative cytotoxic immune cells can directly eliminate iRBCs and the intracellular parasite in an effector:target mechanism. Target iRBCs are labeled to evaluate cell viability, and cocultured with effector cells (CD8+ T, &#947;&#948; T, NK cells, etc.). The lysis percentage is calculated based on tested conditions, compared to a spontaneous lysis control in a flow cytometry-based assay. Ultimately, this killing assay methodology is a major advance in understanding cell-mediated immunity to blood-stage malaria, helping uncover new potential therapeutic targets and accelerate the development of malaria vaccines.

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Here we describe a new method to help elucidate the mechanisms of cellular immunity to Plasmodium during the blood stage of infection. This is an in vitro assay that measures infected red blood cell killing by cytotoxic lymphocytes.&#160;

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective ...

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

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Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes