The authors wish to acknowledge the children and adult plasma donors, and the staff at the Papua New Guinea Institute of Medical Research. The authors would like to thank Amandine B Carmagnac, Catherine Q Nie, Danny W Wilson, Ivo Mueller and Diana S Hansen for their contribution to development of this technique, and thank the Australian Red cross for blood and serum packs. This work was made possible through Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS. This work was supported by National Health and Medical Research Council grants # 1031212 and # 637406, and National Institutes of Health grant # AI089686.

NOTE: All steps, besides centrifugation and flow cytometry, should be performed within a laminar flow hood to maintain sterility. Ensure proper precautions are taken regarding the handling of human samples. The assay is very sensitive for small amounts of plasma. The dilutions provided are optimal for resolving responses ranging from 5 - 78% with plasma from semi-immune children from Papua New Guinea (PNG). The optimal dilution may vary with plasma sets being tested, and therefore it is recommended that plasma be titrated to determine experimental conditions for each application. Ensure the inclusion of 2 negative controls THP-1 cells with merozoites in absence of plasma; and THP-1 cells with merozoites opsonized with a pool of plasma from malaria na&#239;ve individuals. This will allow control for merozoite adherence to THP-1 cells and to enable rigorous flow-cytometry gating of phagocytosis events.
The use of PNG plasma samples was approved by the Medical Research Advisory Committee, Papua New Guinea Ministry of Health, The Walter and Eliza Hall Institute Human Research Ethics Committee (project number 04/04). Written consent was obtained from parents/guardians of all participants.
1. THP-1 Culture
<ol>
	<li>Maintain the human monocytic cell line THP-1 in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS) and 55 &#956;M 2-mercapthoethanol (THP-1 medium) and at 37 &#176;C in a 5% CO2 humidified incubator.</li>
	<li>Maintain cells at a density below 5 x 105 cells/ml. NOTE: Overgrowth will result in differentiation into adherent cells and down-regulation of Fc Receptors. Once cells have been passaged approximately 10 times, thaw a new vial to maintain cell phenotype.</li>
</ol>
2. Antibody Sample Preparation and Dilution
<ol>
	<li>Heat inactivate plasma to be tested and malaria-na&#239;ve control plasma by incubating at 56 &#176;C for 30 min</li>
	<li>Serially dilute plasma samples to 1/2,000 in THP-1 medium.</li>
	<li>Store diluted plasma at -20 &#176;C.</li>
</ol>
3. Culture of Highly Synchronized P. falciparum
NOTE: The GFP-expressing parasite line D10-PfPHG27 was utilized due to its 48 hr life-cycle which assists synchronization of parasites and the controlled timing of E64 addition. Furthermore, because this line expresses GFP in the cytosol, this line allows detection of parasitaemia and free merozoites by flow cytometric detection of GFP. However, the GFP fluorescence intensity is not sufficient to provide visualization within THP-1 cells, and therefore, merozoites are counterstained with Ethidium Bromide (EtBr). Other parasite strains may be utilized, provided that tight synchrony is achievable. Synchronize parasites using a combination of sorbitol treatment to lyse mature forms28 and heparin to inhibit invasion15.
<ol>
	<li>Maintain P. falciparum parasites in O+ human erythrocytes (RBC) at 3% hematocrit in RPMI-1640 medium (pH 7.4) supplemented with 25 mg/ml HEPES, 50 &#956;g/ml hypoxanthine, 10% pooled human serum, 2 mg/ml sodium bicarbonate, and 20 &#956;g/ml gentamycin (Parasite medium). Add 5 mg/ml Blasticidin S-hydrochloride to the culture medium to select for GFP+ parasites.</li>
	<li>Incubate cultures in air tight boxes or alternatively double sealed culture flasks at 37 &#176;C in an atmosphere of 1% O2, 4% CO2 and 95% N2.</li>
	<li>Prepare thin smear slides, fix in 100% methanol for 10 sec, and stain with 10% Giemsa solution in 6.7 mM (pH 7.1) phosphate buffer (Giemsa solution) for 10 min to monitor parasitemia. After staining, rinse slide in water and air-dry. Assess parasitemia using a 100X oil immersion lens.&#160;</li>
	<li>Maintain parasite cultures at a parasitemia below 5% infected RBC by splitting cultures and adding uninfected RBC as required. &#160;</li>
	<li>To synchronize with heparin, add 20 IU/ml of medical-grade heparin to ring-stage cultures.</li>
	<li>When the majority of parasites are at the schizont stage, pellet cells at 300 x g for 5 min, remove heparin-containing medium and resuspend in parasite medium to allow schizont rupture and merozoite invasion.</li>
	<li>After 4 hr, add 20 IU/ml heparin back to cultures, blocking any further merozoite invasion. NOTE: Several cycles of sorbitol and heparin treatment may be required before parasites are sufficiently synchronized. &#160;To generate suitable numbers of merozoites, prepare 150 ml of parasite culture at 3 - 5% parasitaemia.</li>
</ol>
4. Isolation of Late Stage P. falciparum Trophozoites
<ol>
	<li>Thirty-six hr after returning heparin to cultures, pellet cultured cells at 300 x g for 5 min, and resuspend pellet in parasite medium at 25% hematocrit.</li>
	<li>Attach a large magnetic column (matrix volume of 6.3 ml) to a magnet, and equilibrate the column with parasite medium, making sure all air bubbles are removed.</li>
	<li>Add the resuspended P. falciparum culture to the column, and adjust flow rate to one drop per sec.</li>
	<li>Once culture has passed through the column, wash the column with parasite medium until the flow-through runs clear.</li>
	<li>Elute parasites from the column in 30 ml of 37 &#176;C parasite medium.</li>
	<li>Prepare a thin smear of parasites, fix in 100% methanol for 10 sec, and stain with Giemsa solution for 3 min. Examine the slide under the microscope, and ensure that parasites almost fill the red blood cell, and appear to be in early schizont stages. If the parasites have not reached the appropriate maturation stage, return purified parasites to a 37 &#176;C incubator in an atmosphere of 1% O2, 4% CO2 and 95% N2 until suitably developed. NOTE: Purity of greater than 90% infected red blood cells is required to ensure RBCs do not contaminate merozoite preparations.</li>
	<li>Treat isolated schizonts with 10 &#956;M E64 (Epoxysuccinyl-L-leucylamido(4-guanidino)butane (E64) for 6 - 10 hr. NOTE: Longer incubation times with E64 are possible, however the merozoites produced will no longer be invasive.</li>
</ol>
5. Isolation of P. falciparum Merozoites
<ol>
	<li>Following incubation with E64, smear parasites, fix cells in 100% methanol, and stain with Giemsa solution to assess the formation of membrane-enclosed merozoites. NOTE: A good yield of merozoites will be obtained if greater than 50% of parasites have formed membrane enclosed merozoites.</li>
	<li>Pellet E64-treated schizonts at 1,900 x g for 8 min. Wash with 50 ml of RT RPMI-1640 medium (pH 7.4) supplemented with 25 mg/ml HEPES, 50 &#956;g/ml hypoxanthine, 2 mg/ml sodium bicarbonate, and 20 &#956;g/ml gentamycin (wash medium) to remove remaining human serum.</li>
	<li>Resuspend the pellet in 2 ml of wash medium. NOTE: Using FCS-containing THP-1 media will produce frothing during filtration.</li>
	<li>Filter the 2 ml of resuspended E64-schizonts through a 1.2 &#956;m/32 mm syringe filter. Collect the filtrate in a 10 ml tube. NOTE: The filtrate contains merozoites and hemozoin crystals, with un-ruptured schizonts and debris collected in the filter.</li>
	<li>Attach a small magnetic column to a magnet, and equilibrate with 500 &#956;l THP-1 medium.</li>
	<li>Pass the filtrate over the magnetic column. Collect the flow-through. NOTE: Hemozoin will bind to the column, while merozoites will pass through.</li>
	<li>Pass the flow-through over the column two more times to remove hemozoin, collecting the flow through each time.</li>
	<li>Rinse the column with 2 ml of RT THP-1 medium to collect any remaining merozoites.</li>
	<li>Add EtBr at 10 &#956;g/ml (final concentration) and stain for 30 min at RT. Protect from light to prevent photobleaching. Note: Ensure that all EtBr contaminated liquid and plastic waste is disposed of in a manner suitable for cytotoxic chemicals. Merozoites are no longer invasive following this incubation.</li>
	<li>Spin down merozoites at 4,000 x g for 10 min.</li>
	<li>Discard supernatant into appropriate waste container.</li>
	<li>Resuspend merozoite pellet in 4 ml THP-1 medium, and spin down at 4,000 x g for 10&#160;min.</li>
	<li>Add THP-1 medium up to a volume of 15 ml, and protect from light.</li>
</ol>
6. Quantifying Merozoite Concentration by Flow Cytometry
<ol>
	<li>Bring counting beads to RT.</li>
	<li>Prepare PBS with 0.5% BSA for diluting merozoites.</li>
	<li>Count merozoites at 3 dilutions; 1 in 100; 1 in 50 and 1 in 25. Prepare 3 FACS tubes with 940, 930 and 910 &#956;l of PBS+0.5% BSA, respectively. Add 10, 20 and 40 &#956;l of purified merozoites per tube, respectively.</li>
	<li>Vortex counting beads for 30 sec, and add 50 &#956;l of beads per FACS tube containing diluted merozoites.</li>
	<li>Run the three diluted merozoite samples on a flow cytometer equipped with a 488 nm laser. Set a stringent gate for merozoites based on EtBr and GFP fluorescence dual fluorescence, and gate counting beads by fluorescence in a separate channel. Acquire events until 2,000 beads are collected. &#160;Note: These instructions refer to counting GFP+ merozoites that have been counter-stained with EtBr. If not utilizing GFP expressing parasites then set merozoite gates based on side scatter and EtBr fluorescence.</li>
	<li>Determine the ratio of merozoites:beads by dividing the number of merozoite events by the 2,000 counting bead events, calculate a ratio for each dilution. Use the counting bead batch specifications to determine the number of beads in the 50&#160;&#956;l of beads added per tube.&#160;</li>
	<li>Multiply the number of beads in 50&#160;&#956;l by the dilution factor and by the merozoite:bead ratio for that dilution factor. This number equates to the concentration of merozoites per ml. Perform these steps for each dilution.</li>
	<li>Average the merozoite concentrations measured across the three dilutions. NOTE: Standard deviations in excess of 10% for concentrations determined for 1 in 100, 1 in 50, and 1 in 25 dilutions indicate technical inaccuracies in preparing counting samples.</li>
	<li>Resuspend merozoites at 8 x 106 merozoites/ml in THP-1 medium to ensure a final ratio of four merozoites per THP-1 cell. NOTE: Other merozoite to THP-1 ratios may be used, and concentrations should be modified accordingly.</li>
</ol>
7. Phagocytosis Assay
<ol>
	<li>Add 200 &#956;l of heat inactive FCS per well of 96 well U-bottom plates, and leave at RT O/N to block plates. Following incubation, remove FCS and wash wells twice with 200 &#956;l sterile PBS. Remove PBS and store plates at 4 &#176;C until required.</li>
	<li>Calculate the number of THP-1 cells required to test plasma samples and controls, allowing triplicate wells per sample at 1 x 105 THP-1 cells per well.</li>
	<li>Determine THP-1 culture concentration by loading 10&#160;&#181;l of culture on to a haemocytometer slide. Count the number of cells present in 4 sets of 16 corner squares of the haemocytometer under a microscope, then average the counts. Multiply the average count by 10,000 to calculate the number of cells per ml.</li>
	<li>Spin down the required number of THP-1 cells at 500 x g for 5 min, and resuspend at 6.7 x 105 cells/ml in THP-1 medium.</li>
	<li>Add 150&#160;&#956;l of THP-1 cells in THP-1 medium per well to a FCS-blocked 96 well U-bottom plate, resulting in 105 cells/well. Prepare 3 wells per sample that will be tested. Return plate to incubator until ready to add merozoites.</li>
	<li>Add 150 &#956;l of merozoite preparation at 8 x 106/ml per well to a separate FCS-blocked 96 well U-bottom plate. Prepare one well per sample tested</li>
	<li>Add 10 &#956;l of prepared diluted plasma samples per well, to the plate that contains the merozoites, and mix well to ensure homogenous solution.</li>
	<li>Remove the THP-1 plate from the incubator, and add 50 &#956;l of the merozoite/plasma solution per well containing THP-1 cells, with triplicate wells per plasma sample.</li>
	<li>Mix well, and incubate at 37 &#176;C in 5% CO2, humidified incubator for 40 min. Cover with foil to protect from light.</li>
	<li>After 40 min, centrifuge samples for 5 min at 500 x g in a 4 &#176;C prechilled centrifuge to arrest phagocytosis.</li>
	<li>Remove supernatant, and wash cells twice in ice-cold PBS+0.5% BSA+2 mM EDTA (FACS buffer). NOTE: EDTA will facilitate in maintaining a single cell suspension for flow cytometry analysis.</li>
	<li>Fix cells in 90 &#956;l of 2% paraformaldehyde (PFA) in PBS+0.5% BSA+2 mM EDTA (FACS fixative). Leave on ice until acquisition, protected from light.</li>
</ol>
8. Flow Cytometry
<ol>
	<li>Acquire samples using a flow cytometer equipped with a 488 nm laser and high throughput plate reader attachment. NOTE: This will enable up to 400 plasma samples to be tested from one merozoite preparation. Cells can also be transferred to tubes for manual sample loading.</li>
	<li>Gate viable THP-1 cells by forward and side scatter.</li>
	<li>Set the EtBr positive gate based on the &#8216;THP-1 cells with merozoites and no plasma&#8217; control. &#160;</li>
	<li>Acquire a minimum of 10,000 events per sample.</li>
</ol>
9. Data Analysis
<ol>
	<li>Analyze data using flow cytometry analysis software, and set stringent gates for EtBr positive events.</li>
	<li>Calculate the percentage phagocytosis for each sample: the % of EtBr+ cells for the sample minus the % positive cells with non-immune plasma.</li>
	<li>Average results across triplicates. NOTE: Standard deviations for a triplicate in excess of 10% indicate experimental inaccuracies. Some background EtBr positive events may be observed as a result of merozoite adhesion to the surface of THP-1 cells, but should be consistent across all samples containing merozoites. Setting the gate based on the &#8216;THP-1 cells with merozoites and no plasma&#8217; control, and then subtracting any background of the &#8216;THP-1 cells with non-immune plasma&#8217; control will result in % phagocytosis that reflects the fluorescence due to internalized/phagocytosed merozoites only.</li>
</ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+fluorescent+Plasmodium+falciparum+for+in+vitro+growth+inhibition+assays.">Development of fluorescent Plasmodium falciparum for in vitro growth inhibition assays.</a> Malar J. 9, 152 (2010).</li><li>Lambros, C., Vanderberg, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronization+of+Plasmodium+falciparum+erythrocytic+stages+in+culture.">Synchronization of Plasmodium falciparum erythrocytic stages in culture.</a> J Parasitol. 65, 418-420 (1979).</li><li>Braun-Breton, C., Rosenberry, T. L., da Silva, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+the+proteolytic+activity+of+a+membrane+protein+in+Plasmodium+falciparum+by+phosphatidyl+inositol-specific+phospholipase.">Induction of the proteolytic activity of a membrane protein in Plasmodium falciparum by phosphatidyl inositol-specific phospholipase.</a> C. Nature. 332, 457-459 (1988).</li><li>Jaramillo, M., Godbout, M., Olivier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemozoin+induces+macrophage+chemokine+expression+through+oxidative+stress-dependent+and+-independent+mechanisms.">Hemozoin induces macrophage chemokine expression through oxidative stress-dependent and -independent mechanisms.</a> J Immunol. 174, 475-484 (2005).</li><li>Schofield, L., Tachado, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+host+cell+function+by+glycosylphosphatidylinositols+of+the+parasitic+protozoa.">Regulation of host cell function by glycosylphosphatidylinositols of the parasitic protozoa.</a> Immunology and cell biology. 74, 555-563 (1996).</li><li>Khusmith, S., Druilhe, P., Gentilini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+Plasmodium+falciparum+merozoite+phagocytosis+by+monocytes+from+immune+individuals.">Enhanced Plasmodium falciparum merozoite phagocytosis by monocytes from immune individuals.</a> Infection and immunity. 35, 874-879 (1982).</li><li>Lunov, O., 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=Differential+uptake+of+functionalized+polystyrene+nanoparticles+by+human+macrophages+and+a+monocytic+cell+line.">Differential uptake of functionalized polystyrene nanoparticles by human macrophages and a monocytic cell line.</a> ACS. 5, 1657-1669 (2011).</li><li>Tebo, A. E., Kremsner, P. G., Luty, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fcgamma+receptor-mediated+phagocytosis+of+Plasmodium+falciparum-infected+erythrocytes+in+vitro.">Fcgamma receptor-mediated phagocytosis of Plasmodium falciparum-infected erythrocytes in vitro.</a> Clinical and experimental immunology. 130, 300-306 (2002).</li><li>McGilvray, I. D., Serghides, L., Kapus, A., Rotstein, O. D., Kain, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonopsonic+monocyte/macrophage+phagocytosis+of+Plasmodium+falciparum-parasitized+erythrocytes:+a+role+for+CD36+in+malarial+clearance.">Nonopsonic monocyte/macrophage phagocytosis of Plasmodium falciparum-parasitized erythrocytes: a role for CD36 in malarial clearance.</a> Blood. 96, 3231-3240 (2000).</li></ol>

The authors have nothing to disclose.

To measure merozoite phagocytosis, proficiency in two techniques is required: purification of merozoites and THP-1 phagocytosis assay. The most critical steps for combining these two techniques are: 1) Highly synchronized parasites; 2) Adding E64 at the correct time to yield membrane enclosed merozoites; 3) Removing hemozoin to avoid aggregates; 4) Accurate merozoite counting by flow cytometry; 5) the dilution of plasma used; and 6) maintaining low cell density and passage number of THP-1 cells. Careful consideration of these key aspects will ensure robust phagocytosis responses are observed.
Although, described here is the preparation of merozoites for assessing phagocytosis, the technique can be utilized for a wide range of applications. Irrespective of the down stream methodology, optimal merozoite preparations depend on correctly timing E64 addition in order to generate merozoites. The E64 method described here has been shown to yield invasive merozoites for use in high resolution microscopy of invasion events and drug sensitivity assays15-20. While merozoite viability is not essential for phagocytosis, integrity of the merozoite surface coat is required. Therefore the E64 method described here allows high quality merozoites to be produced for assessing antibody responses to the surface coat. As outlined in Figure 1, if E64 is added too early or too late membrane enclosed merozoites are not formed. For this reason, highly synchronous parasite cultures are required. Here, the use of sorbitol and heparin treatments to tightly synchronize D10-GFP parasite cultures to a window of 2 hr is described. Heparin can promote gametocytogenesis in other lab isolates, and hence should be used carefully. Alternative synchronization methods such as alanine may also be used, provided that tightly synchronized parasites are produced29. If E64 is added to asynchronous parasites, a lower proportion of membrane-enclosed merozoites will be produced and remaining parasite-infected RBC will either rupture normally or develop abnormal morphology. The presence of parasites that have not developed into membrane enclosed merozoites will result in clogging of the filter and significantly reduce the merozoite yield obtained.&#160;
During filtration of membrane-enclosed merozoites, hemozoin is liberated from the digestive vacuoles and is present as free crystals in solution. Hemozoin is highly proinflammatory and has been reported to modulate monocyte and macrophage phagocytosis responses30,31 . In addition to modulating phagocytosis, as shown in Figure 2, hemozoin can form aggregates with merozoites in solution. As THP-1 cells can phagocytose these aggregates, this can be a major confounder for the resolution of antibody mediated phagocytosis. Therefore, removal of hemozoin is necessary in this assay to avoid additional complexity of hemozoin on phagocyte biology. In addition, failure to remove hemozoin may also be deleterious when using this technique to generate free merozoites for other applications, especially where merozoite quantification is required.
As outlined in Figure 4, the number of merozoites added to the assay can influence the degree of phagocytosis by THP-1 cells. Although flow cytometry allows for enumeration of merozoite concentration, careful pipetting and replicate counts of merozoites are necessary to improve accuracy. This is especially critical if multiple parasite lines are to be tested side-by-side. It has previously been demonstrated that plasma from semi-immune children from PNG can be significantly diluted before responses decline23. Described here is the optimal dilution of plasma (1/120,000 final dilution of plasma) for a cohort of 5 - 12 year old PNG children that produced the large range of phagocytosis responses described in Figure 5. This range allowed for stratification of responses into four groups (0 - 19%, 20 - 39%, 40 - 59% and 60 - 79% phagocytosis), and regression modeling revealed that opsonizing responses were associated with protection from clinical disease and high-density parasitaemia10 For different cohort studies it may be necessary to adjust plasma dilution to ensure the phagocytosis by THP-1 cells is not saturated or below the level of detection.
Flow cytometry allows fast and quantifiable phagocytosis with improved accuracy over microscopy. This protocol is a high throughput, plate based and automated acquisition of 96 well plates to study naturally acquired humoral immunity. The method requires staining of merozoites with ethidium bromide, however alternative DNA stains such as SYBRgreen, DAPI and propidium iodine, membrane stains or protein stains could be utilized. This assay would be amenable to primary monocytes or neutrophils, and could be adapted if phagocyte or FcR biology was of interest. Primary cells or THP-1 cells differentiated in vitro with PMA can be used to study phagocytosis in malaria and other pathogens12,32-34. However using primary cells may be challenging as these cells are adherent and also display non-antibody mediated phagocytosis35. In addition, variability in purity, viability and functionality are some key limitations to the use of primary phagocytic cells. The Fc receptors involved in merozoite phagocytosis remain uncharacterized, and therefore using blocking antibodies to specific Fc Receptors could elucidate the contribution of each Fc Receptor to merozoite phagocytosis. As THP-1 phagocytosis is FcR dependent, this enables straightforward interpretation of the phagocytosis observed. This assay also lends itself to addressing the antigen specificity of opsonizing antibodies which could be achieved by utilizing knock-out parasites for merozoite surface antigens, or using affinity purified human antibodies to merozoite surface proteins. Furthermore, in depth studies of cytokine responses following merozoite phagocytosis are lacking, and could be achieved using this assay.
These two techniques constitute considerable advances to the study of functional antimerozoite antibodies. The purification of high quality merozoites is advantageous to using cryopreserved merozoites, or fluorescent microspheres coated with merozoite antigens. Although this assay has been used as a tool for evaluating naturally acquired immunity, it may prove an important tool for addressing the acquisition of immunity in response to vaccination. While it has been recently shown that the phagocytosis or opsonised merozoites is associated with protection from clinical malaria, it is not possible to draw direct conclusions from in vitro THP-1 phagocytosis to in vivo phagocytosis of merozoites as phagocytosis in this assay occurs under static conditions and in the absence of competing red blood cells. The potential adaptations of this assay may thus provide a versatile tool for further understanding of malaria and merozoite phagocytosis.

The importance of antibodies for immunity&#160; to Plasmodium falciparum malaria was shown 40 years ago, when immunoglobulin from hyperimmune adults was passively transferred to children suffering from severe malaria resulting in alleviated disease1. Consequently, considerable effort has sought to identify targets of protective malarial immunity, mostly through measuring antibody titers&#160;to peptides or bacterially expressed proteins by ELISA. ELISA based serology has also proven highly variable between studies, and does not address antibody functionality2. Many malaria antigens induce a cytophilic IgG1 and IgG3 antibody profile, particularly merozoite surface antigens3. This subclass bias suggests that antibody-Fc-receptor (FcR) interactions with phagocytes are important for the effector functions of opsonizing antimerozoite antibodies4. Several merozoite antigen vaccines under development are designed to elicit phagocyte effector functions5,6 and although significant evidence for the importance of antibody-FcR interactions in models of rodent malaria exists7-9, and a few recent studies support the importance of functional antibodies and phagocyte effector functions for immunity to malaria in humans10,11, this area remains poorly studied. Study into merozoite specific opsonizing antibodies has been limited by two factors; the difficulty in isolating good quality merozoites; and variable phagocytosis responses from primary cells.
Until recently, high speed centrifugation or Percoll density gradients were utilized to isolate merozoites from culture supernatants of rupturing schizont cultures. These merozoites were rarely viable, and often further manipulated by density centrifugation and multiple wash steps12, or cryopreservation11 before use in assays. These processes potentially detach many peripherally associated proteins from the merozoite surface, proteins known to be antigenic targets of malarial immunity13. Recently the cysteine protease inhibitor trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane (E64) has been used to generate viable merozoites. E64 prevents schizont rupture, generating membrane enclosed merozoites14, which can be disrupted by filtration to liberate viable merozoites15,16. This technique has lead to the spatial resolution of numerous proteins during erythrocyte invasion15,17-19 and has clarified the stage specific effect of several antimalarial drugs16,20. However, the generation of viable merozoites remains technically challenging. To aid in the dissemination of this technique and application to functional assays of immunity, a detailed protocol for viable merozoite purification and their use in a standardized functional assay of antibody:cellular cooperation in opsonization and phagocytosis is described here.
This technique demonstrates a significant advance over previous in vitro assays of merozoite opsonization, such as neutrophil respiratory burst, Antibody Dependent Cellular Inhibition (ADCI), and alternative merozoite phagocytosis assays. These assays are poorly reproducible due to variation in parasite inputs and the activity of primary phagocytic cells11,21. Contaminating hemozoin may also profoundly affect phagocyte function22. A recently reported robust and reproducible merozoite phagocytosis assay23 utilizes the promonocytic cell line THP-124. This is an ideal cell type for high throughput flow cytometry assays as it is non-adherent and specifically performs Fc-Receptor mediated phagocytosis25,26. An additional complexity while investigating phagocytosis is that the degree of phagocytosis is dependent on the number of merozoites relative to THP-1 cells and plasma concentration. To ensure reproducibility between experiments, merozoites should be enumerated and a defined concentration used. Due to their small size, flow cytometric quantification is required.
The procedure described here removes hemozoin and generates viable merozoites, and describes an application of these merozoites for flow cytometric enumeration of merozoites followed by opsonization and phagocytosis. Although technically demanding, the techniques described may prove useful in elucidating the contribution of merozoite surface specific antibody responses to naturally acquired and vaccine induced immunity.

<table border="0" cellpadding="0" cellspacing="0" width="1034">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:317px;">THP-1 cell line</td>
			<td style="width:225px;">American Type Culture Collection</td>
			<td style="width:119px;">TIB-202</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">RPMI-1640</td>
			<td>Gibco</td>
			<td style="width:119px;">31800-089</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Fetal Calf Serum (FCS)</td>
			<td style="width:225px;">Invitrogen</td>
			<td style="width:119px;">10099-141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-mercapthoethanol&#160;</td>
			<td style="width:225px;">Sigma-Aldrich</td>
			<td>M6250-100ML</td>
			<td>Use in Fume Hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Pen/Strep Solution (100x)</td>
			<td style="width:225px;">Sigma</td>
			<td style="width:119px;">P0781</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">HEPES</td>
			<td style="width:225px;">SAFC</td>
			<td style="width:119px;">90909C</td>
			<td style="width:373px;">Cell culture grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hypoxanthine</td>
			<td style="width:225px;">Calbiochem</td>
			<td style="width:119px;">4010</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Human Serum&#160;</td>
			<td style="width:225px;">Donation from Autralian Red Cross</td>
			<td style="width:119px;"></td>
			<td>Available commercially (i.e. Invitrogen)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium bicarbonate (NaHCO3)</td>
			<td style="width:225px;">Merck</td>
			<td style="width:119px;">1.06329.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gentamycin</td>
			<td style="width:225px;">Pfizer</td>
			<td style="width:119px;">61022027</td>
			<td style="width:373px;">Injection Quality</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">heparin Sodium BP (5000IU/mL)</td>
			<td style="width:225px;">Pfizer</td>
			<td style="width:119px;"></td>
			<td>procine origin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blasticidin S-hydrochloride</td>
			<td style="width:225px;">Sigma-Aldrich</td>
			<td>15205</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-sorbitol</td>
			<td style="width:225px;">Sigma-Aldrich</td>
			<td style="width:119px;">&#160;50-70-4&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">E64 Protease Inhibitor</td>
			<td style="width:225px;">Sigma-Aldrich</td>
			<td>E3132-10MG</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">KH2PO4</td>
			<td style="width:225px;">Sigma-Aldrich</td>
			<td style="width:119px;">98281-100G</td>
			<td>Use for phosphate buffer</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2HPO4.2H20</td>
			<td style="width:225px;">Merck&#160;</td>
			<td style="width:119px;">10383.4G</td>
			<td>Use for phosphate buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:317px;">Giemsa&#39;s azur eosin methylene blue solution</td>
			<td style="width:225px;">Merck&#160;</td>
			<td style="width:119px;">1.09204.0500</td>
			<td style="width:373px;">1:10 dilution in 6.7mN Phosphate buffer (make fresh each stain)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">EDTA disodium salt</td>
			<td style="width:225px;">Merck</td>
			<td style="width:119px;">10093.5V</td>
			<td style="width:373px;">0.1M, pH 7.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acrodisc Syringe Filters 1.2-&#956;m/32-mm</td>
			<td style="width:225px;">Pall Life Sciences</td>
			<td style="width:119px;">4656</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">QuadroMACS Separator (for small column)</td>
			<td style="width:225px;">MACS Miltenyi BioTec</td>
			<td>130-091-051</td>
			<td>Interchangeable with MidiMACS&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">VarioMACS Separator (for large columns)</td>
			<td style="width:225px;">MACS Miltenyi BioTec</td>
			<td>130-090-282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">MACS MultiStand</td>
			<td style="width:225px;">MACS Miltenyi BioTec</td>
			<td style="width:119px;">130-042-303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LS Column (small magnetic column)</td>
			<td style="width:225px;">MACS Miltenyi BioTec</td>
			<td style="width:119px;">130-042-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">large magnetic column</td>
			<td style="width:225px;">MACS Miltenyi BioTec</td>
			<td>130-041-305</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">Ethidium Bromide</td>
			<td style="width:225px;">Bio-Rad</td>
			<td style="width:119px;">1510433</td>
			<td>Cytotoxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CountBright Absolute Counting Beads</td>
			<td style="width:225px;">Invitogen</td>
			<td>C36950</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:317px;">BD FACSCalibur Flow Cytometer with HTS plate reader</td>
			<td style="width:225px;">BD Biopsciences</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">EDTA disodium salt</td>
			<td style="width:225px;">Merck</td>
			<td style="width:119px;">10093.5V</td>
			<td style="width:373px;">0.1M, pH 7.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:317px;">FlowJo cytometry analysis software</td>
			<td style="width:225px;">Tree Star</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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;

The maturation stage of parasites prior to E64 treatment is critical for generating membrane-enclosed merozoites. Figure 1Ai shows the appropriate maturation stage of schizonts for adding E64. The parasites should be large and almost fill the red blood cell. A dappled appearance of Giemsa stain indicates merogony has commenced, and E64 should be added to yield membrane-enclosed merozoites (Figure 1Aii). If E64 is added earlier to trophozoite stage parasites, membrane enclosed merozoites are not generated even after 12 hr of E64. Instead parasites take on abnormal morphology such as enlargement of the digestive vacuole (Figure 1Bi and 1Bii), and merozoites are not formed. If E64 is added later to schizonts, membrane-enclosed merozoites are not generated as schizont rupture is uninhibited (Figure 1Ci and 1Cii). A high level of parasite synchrony is required, otherwise a range of all three outcomes described will be seen after E64 treatment.
Removal of hemozoin is another critical step for purifying merozoites. If merozoites are not purified from hemozoin then merozoite-hemozoin clusters form which cannot be dislodged by pipetting. These aggregates run on the cytometer as single events, albeit with different forward-scatter and side-scatter characteristics than single merozoites, resulting in inaccurate merozoite counting by flow cytometry (Figure 2A). As THP-1 cells can also phagocytose hemozoin, these merozoite-hemozoin clusters can also be phagocytosed (Figure 2B). These aggregates are highly EtBr fluorescent as they contain multiple merozoites. Phagocytosis of aggregates results in a THP-1 EtBr fluorescence profile equivalent to that observed for the phagocytosis of multiple individual merozoites. Hence, if hemozoin is not removed, the EtBr fluorescence of THP-1 cells will be an overestimate of the opsonizing potential for the plasma being tested. Hemozoin is also reported to significantly alter phagocyte function. GFP positive merozoites are counter stained with EtBr to increase visualization of merozoites phagocytosed by THP-1 cells. Using the conditions described in this protocol, all GFP positive merozoites are counterstained with EtBr which has a brighter fluorescence intensity (Figure 3A). Phagocytosis assessment by EtBr fluorescence enables superior resolution of merozoite phagocytosis than GFP fluorescence (Figure 3B).
The number of merozoites added to the assay will modulate the amount of phagocytosis observed. For this reason, accurate counting by flow cytometry is critical. Increasing ratios of merozoites:THP-1 will result in increased adherence of merozoites to THP-1 cells in the absence of plasma (Figure 4A). Increasing merozoite:THP-1 ratios also results in increased phagocytosis responses when merozoites are opsonized (Figure 4B). A 4:1 merozoite:THP-1 ratio is recommended for robust phagocytic responses. Using this ratio, and the dilution of plasma indicated, this assay is able to resolve low, intermediate and high levels of opsonizing antibodies. Between 0 and 78 percent phagocytosis responses have been observed using PNG plasma samples and the other conditions described. Such responses are recently shown to associate with naturally acquired clinical immunity to malaria10. Figure 5 shows examples of the 4 quartiles of phagocytosis responses from PNG individuals.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51590/51590fig1highres.jpg" width="500" /> 
Figure 1. Timing of E64 addition is critical for generating membrane-enclosed merozoites. (A) Appropriate maturation stage of parasites i) prior to E64 treatment, and ii) membrane-enclosed merozoites produced after 6 hr of E64 treatment. (B) membrane enclosed merozoites are not generated if E64 is added to immature parasites; i) Late stage trophozoites, and ii) after 12 hr of E64. (C) Schizont rupture is not inhibited if E64 is added to late segmented schizonts; i) Late stage schizonts, and ii) after 6 hr of E64. Scale bar represents 10 &#956;m.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51590/51590fig2highres.jpg" width="500" /> 
Figure 2. Hemozoin removal is critical to generate a single-cell merozoite suspension. Merozoite-hemozoin aggregates form following filtration of membrane enclosed merozoites, unless hemozoin is magnetically separated from merozoites. (A) Forward-scatter and side-scatter plots of merozoites with hemozoin removed and hemozoin retained. (B) Hemozoin-merozoite aggregates can be phagocytosed by THP-1 cells. Diff-Quick stained cyto-spin slides of THP-1 cells incubated with PNG plasma opsonized merozoite preparations with or without hemozoin. Scale bar represents 10 &#956;m.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51590/51590fig3highres.jpg" width="500" /> 
Figure 3. Ethidium Bromide staining allows for superior resolution of merozoite phagocytosis. D10-GFP purified merozoites are counterstained with EtBr to improve fluorescence intensity. (A) Flow cytometry histrogram of purified merozoites with gating on GFP positive merozoites, and a dot blot showing EtBr fluorescence within this gated GFP positive population. (B) Forward scatter versus GFP and forward scatter versus EtBr of THP-1 cells following phagocytosis of GFP and EtBr dual fluorescent merozoites.&#160; Gates were drawn based on the THP-1 cells with merozoites and no plasma control.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51590/51590fig4highres.jpg" width="500" /> 
Figure 4.&#160;The merozoite:THP-1 ratio influences the degree of phagocytosis observed. (A) Five merozoite:THP-1 ratios are depicted; 50:1, 20:1, 10:1, 4:1, 1:1. Cells only control indicates background fluorescence due to THP-1 cells. THP-1 EtBr fluorescence for each ratio is indicated for merozoites opsonized with a pool of PNG plasma or with nonimmune Australian plasma. (B) Mean fluorescence intensity of THP-1 cells for different merozoite:THP-1 ratios, and opsonization with a pool of PNG plasma or nonimmune Australian plasma (mean+SEM). <a href="https://www.jove.com/files/ftp_upload/51590/51590fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/51590/51590fig5highres.jpg" width="500" /> 
Figure 5. A large range of opsonizing antibodies can be measured. Merozoites were opsonized with plasma from PNG individuals and incubated with THP-1 cells. Four representative phagocytosis responses are shown. Gates were drawn based on the THP-1 cells with merozoites and no plasma control, and numbers indicate the %phagocytosis after subtraction of the THP-1 cells with non-immune plasma control. <a href="https://www.jove.com/files/ftp_upload/51590/51590fig5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays at JoVE.com

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.

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 ...

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Research

JoVE Journal

Immunology and Infection

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5). Sequestration is also found in most microvascular tissues of the human body2, 3. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood.
The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo.
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7.

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules 1. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes 2 and restrict their environment to a more favorable low oxygen pressure 3. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.
Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria 4,5. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor 6. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)7,8 has also been associated with severe disease 9.
One of the most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps 10. Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM 11. In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian 12 and Mozambican patients 13, (although not in Malian 14).
With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay 15. Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully ...

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the &asymp;40% limit, the investigator usually keeps the percentage at around 10%. In many cases it is necessary to isolate the parasite-containing red blood cells (RBCs) from the uninfected ones, to enrich the culture and proceed with a given experiment. 
When P. falciparum infects the erythrocyte, the parasite degrades and feeds from haemoglobin 2, 3. However, the parasite must deal with a very toxic iron-containing haem moiety 4, 5. The parasite eludes its toxicity by transforming the haem into an inert crystal polymer called haemozoin 6, 7. This iron-containing molecule is stored in its food vacuole and the metal in it has an oxidative state which differs from the one in haem 8. The ferric state of iron in the haemozoin confers on it a paramagnetic property absent in uninfected erythrocytes. As the invading parasite reaches maturity, the content of haemozoin also increases 9, which bestows even more paramagnetism on the latest stages of P. falciparum inside the erythrocyte.
Based on this paramagnetic property, the latest stages of P. falciparum infected-red blood cells can be separated by passing the culture through a column containing magnetic beads. These beads become magnetic when the columns containing them are placed on a magnet holder. Infected RBCs, due to their paramagnetism, will then be trapped inside the column, while the flow-through will contain, for the most part, uninfected erythrocytes and those containing early stages of the parasite.
Here, we describe the methodology to enrich the population of late stage parasites with magnetic columns, which maintains good parasite viability 10. After performing this procedure, the unattached culture can be returned to an incubator to allow the remaining parasites to continue growing.

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

The paramagnetic properties of hemozoin are used to isolate late stages of Plasmodium falciparum-infected red blood cells growing in culture. The method is simple and fast and does not affect the subsequent invasive capabilities of the parasites.

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the ...

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The molecular basis of this resistance, which is known to be multifactorial, remains incompletely understood. Recent studies found that the differential expression of erythrocyte microRNAs, once translocated into malaria parasites, affect both gene regulation and parasite growth. These miRNAs were later shown to inhibit mRNA translation by forming a chimeric RNA transcript via 5&#39; RNA fusion with discreet subsets of parasite mRNAs. Here, the techniques that were used to study the functional role and putative mechanism underlying erythrocyte microRNAs on the gene regulation and translational potential of P. falciparum, including the transfection of modified synthetic microRNAs into host erythrocytes, will be detailed.&#160; Finally, a polysome gradient method is used to determine the extent of translation of these transcripts. Together, these techniques allowed us to demonstrate that the dysregulated levels of erythrocyte microRNAs contribute to cell-intrinsic malaria resistance of sickle erythrocytes.

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Human microRNAs translocate from host erythrocytes to Plasmodium falciparum parasites. Here, the techniques used to transfect synthetic microRNAs into host erythrocytes and isolate all RNAs from P. falciparum are described. In addition, this paper will detail a method of polysome isolation in P. falciparum to determine the ribosomal occupancy and translational potential of parasite transcripts.

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections.
Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring.
In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells.
In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular bacteria based on the GFP signal, with only intracellular bacteria being able to express GFP. This allows the robust detection of single intracellular bacteria before intracellular proliferation is initiated.

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols.

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

Electrochemiluminescence Assays for Human Islet Autoantibodies

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ECL assay is a nonradioactive fluid phase assay for islet autoantibodies with higher sensitivity and specificity than the current 'gold' standard radio-binding assay (RBA). ECL assays can more precisely define the onset of presymptomatic T1D by distinguishing the high-risk, high-affinity autoantibodies from the low-risk, low-affinity autoantibodies generated in RBAs, and conventional enzyme-linked immunosorbent assays (ELISA). The antigen protein used in this ECL assay is labeled with Sulfo-tag and Biotin, respectively. Each ECL autoantibody assay that uses a particular antigen protein needs an optimization step before it can be used for laboratory application. This step is especially vital in determining the requirements for serum acid treatments, concentrations, and ratios of the two different antigens labeled with Sulfo-tag and Biotin. To perform the assay, serum samples are mixed with Sulfo-tag-conjugated and biotinylated capture antigen protein in phosphate buffered solution (PBS), containing 5% Bovine Serum Albumin (BSA). Afterwards, the samples are incubated overnight at 4 &#176;C. The same day, a streptavidin-coated plate is prepared with blocker buffer and incubated overnight at 4 &#176;C. On the second day, wash the streptavidin plate and transfer the serum-antigen mixture onto the plate. Place the plate on the plate shaker, set it at low speed, and incubate at room temperature for 1 h. Subsequently, the plate is washed again, and reader buffer is added. The plate is then counted on the plate reader machine. The results are conveyed through an index, which is generated from internal standard positive and negative control serum samples.

Here, we presented the methods to detect human islet autoantibodies using electrochemiluminescence (ECL) assays. The protocol, used to predict type 1 diabetes, can be expanded upon to detect autoantibodies for other autoimmune diseases.

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ...

Purification of a High Molecular Mass Protein in Streptococcus mutans

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has been disrupted. Loss of function following gene disruption is subsequently restored by exogenous addition of the product of the disrupted gene. This helps to determine the function of the gene. A method previously described involves generating a gtfC gene-disrupted Streptococcus mutans strain. Here, an undemanding method is described for purifying the gtfC gene product from the newly generated S. mutans strain following the gene disruption. It involves the addition of a polyhistidine-coding sequence at the 3&#8242; end of the gene of interest, which allows simple purification of the gene product using immobilized metal affinity chromatography. No enzymatic reactions other than PCR are required for the genetic modification in this method. The restoration of the gene product by exogenous addition after gene disruption is an efficient method for determining gene function, which may also be adapted to different species.

Purification of a High Molecular Mass Protein in Streptococcus mutans

Described here is a simple method for the purification of a gene product in Streptococcus mutans. This technique may be advantageous in the purification of proteins, especially membrane proteins and high molecular mass proteins, and can be used with various other bacterial species.

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has ...