Maiken Visti is thanked for excellent technical assistance. This work was partly funded by a grant (MAVARECA II; 17-02-KU) from the Ministry of Foreign Affairs of Denmark and administered by Danida Fellowship Centre. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The human serum samples used for the results presented here were collected in a separate study19. Collection was approved by the Institutional Review Board of Noguchi Memorial Institute for Medical Research, University of Ghana (study 038/10-11), and by the Regional Research Ethics Committees, Capital Region of Denmark (protocol H-4-2013-083).
1. Parasite culture
NOTE: Follow the local regulations for human pathogens handling.
<ol>
	<li>Maintain P. falciparum parasites according to the standard protocol as described before20 in parasite culture medium (see Table of Materials).</li>
	<li>Keep the parasites tightly synchronized using sorbitol treatment as previously described21.</li>
	<li>To ensure VAR2CSA expression on the surface of the IE, perform repeated rounds of immune-magnetic selection using an anti-VAR2CSA antibody (e.g., PAM1.4 antibody, a cross-reactive human monoclonal VAR2CSA-specific IgG22) coupled to protein G-magnetic beads23.
	<ol>
		<li>In brief, incubate late-stage trophozoite-IEs with magnetic beads coupled to an anti-VAR2CSA antibody and positively select using a magnet.</li>
		<li>Expand the selected parasites in culture for a few cycles until parasitemia is at least 5%.</li>
		<li>Alternatively, select parasites that bind to plastic-immobilized CSA (the receptor for VAR2CSA) as previously described24.</li>
	</ol>
	</li>
	<li>To verify VAR2CSA expression on the selected parasites perform flow cytometry as previously described25.
	<ol>
		<li>In brief, label the late-stage trophozoite-IEs with the same antibody used for the magnetic selection (step 1.3) followed by a fluorescently labeled secondary antibody.</li>
		<li>Measure the IE surface reactivity by flow cytometry. Successful antibody selection normally results in a parasite population that expresses VAR2CSA in most of the parasites (&#8776;80%).</li>
	</ol>
	</li>
	<li>For the phagocytosis assay, use purified mid- to late-stage trophozoite-IEs. Perform purification using magnetic separation as previously described26. 
	NOTE: To accurately determine the stage of the parasites, perform Giemsa staining on blood smears followed by light microscopy observation. Follow standard procedures and morphological guidelines as described before27. Late-stage purification can also be performed using density gradient medium. The IE yield, however, in our experience is lower and, therefore, magnetic purification is preferable.
	<ol>
		<li>For magnetic separation, spin the parasite culture (5-10% parasitemia) at 500 x g for 10 min at room temperature. Remove the supernatant and re-suspend the cell pellet in 10 mL of parasite culture medium.</li>
		<li>Add the parasite suspension into a CS-column coupled to a magnet (see Table of Materials) and let it slowly pass through the column. Trophozoite-IEs are paramagnetic (due to the presence of hemozoin) and will stay into the column mesh.</li>
		<li>Wash the column with 50 mL of parasite culture medium and elute the IEs from the magnetic column using 50 mL of parasite culture medium.</li>
	</ol>
	</li>
	<li>Spin the eluate from 1.5.3 at 500 x g for 10 min at room temperature. Carefully discard the supernatant and re-suspend in 1 mL of parasite culture medium.</li>
	<li>Using a hemocytometer, count the number of IEs (easily identified by light microscopy as erythrocytes with dark hemozoin pigment) and total cell number to calculate the final parasitemia (percentage of IEs). 
	NOTE: Only use parasites with at least&#160;80% purity (80%&#160;IEs).</li>
	<li>Based on the number of serum/antibody samples planned for testing, estimate the total amount of IEs needed and scale up the parasite cultures accordingly. A 75 cm2 culture flask with 25 mL of culture at 5% hematocrit and 5-10% parasitemia should yield enough IEs to run a full 96 well plate. For a full plate (42 samples plus 6 controls in duplicate), a minimum of 1.5 mL parasite suspension at 3.3 x 107 IEs/mL are needed.</li>
</ol>
2. THP-1 cells
NOTE: The THP-1 cell line is used in this assay. This monocyte cell line is derived from a patient with monocytic leukemia28 and can be purchased from ATCC. Maintain the cell line according to the provider&#8217;s instructions in THP-1 culture medium (see Table of Materials).
<ol>
	<li>For regular maintenance, start THP-1 cell culture at 2-4 x 105 cells/mL and subculture when cell concentration reaches 8 x 105 cells/mL. 
	NOTE: Do not allow the cell concentration to exceed 1 x 106 cells/mL and keep track of passage number. Avoid the use of cells that are beyond passage 25. The average doubling time of the THP-1 cell line varies between 19-50 h. Determine the doubling time of the cell batch before starting the phagocytosis experiments. This will help to roughly estimate the necessary amount of culture needed for a determined number of serum samples to be tested (e.g., for a full 96 well plate, 10 mL of culture at 5 x 105 cells/mL are needed).</li>
	<li>Periodically check the THP-1 cells for the surface expression of the Fc&#947;-receptors I (CD64), II (CD32) and III (CD16) as previously described15.
	<ol>
		<li>In brief, stain the THP-1 cells (separately) with anti-CD64, anti-CD32 and anti-CD16 fluorescently labeled antibodies (Table of Materials) for 30 min at room temperature (1:100 dilution prepared in 2% FBS in PBS).</li>
		<li>Wash three times with 2% FBS in PBS and measure surface staining by flow cytometry. 
		NOTE: The THP-1 cells are negative for CD16 and positive for CD32 and CD64 as previously reported29,30 (Figure 1).</li>
	</ol>
	</li>
	<li>For the phagocytosis assay, set up a THP-1 cell culture flask with 2.5 x 105 cells/mL the day before the experiment to yield around 5 x 105 cells/mL on the day of assay. 
	NOTE: Ensure 4-6 x 105 cells/mL are present on the day of the assay.</li>
</ol>
3. Phagocytosis assay (Figure S1)
<ol>
	<li>Before starting the assay, block (&#62;1 h) two round-bottom 96 well plates (one for the opsonization and another for the phagocytosis) using 150 &#956;L per well of sterile 2% FBS in PBS. 
	NOTE: Label plate 1 as opsonization plate and use it both for ethidium bromide (EtBr) staining and opsonization. Label plate 2 as phagocytosis plate and use both for THP-1 cell plating and for the phagocytosis step.</li>
	<li>Parasite staining and opsonization
	<ol>
		<li>Take the IE suspension prepared in 1.6 and adjust cell count to 3.3 x 107 IEs/mL by adding parasite culture medium and EtBr to achieve a final concentration of 2.5 &#956;g/mL (1:40 dilution, from a 0.1 mg/mL stock). 
		NOTE: EtBr stains the parasite DNA, allowing detection by flow cytometry. 
		CAUTION: EtBr is a mutagen and skin, eye, and respiratory irritant. Use appropriate protection and dispose waste according to local regulations.</li>
		<li>Remove the blocking solution from one of the 96 well plates (opsonization plate), flicking the plate and removing liquid excess over a piece of towel paper.</li>
		<li>Add 30 &#956;L per well of the IE suspension prepared above in the upper half of the plate. Leave one well empty and add 30 &#181;L of parasite culture medium (without IEs for the THP-1 alone control). (Figure S2A). Incubate for 10 min at room temperature and protected from light.</li>
		<li>Add 170 &#181;L per well of parasite culture medium. Spin the plate at 500 x g for 3 min at room temperature&#160;and remove the supernatant by flicking the plate over an appropriate waste container.</li>
		<li>Wash the EtBr-labeled IEs two more times using 200 &#181;L per well of parasite culture medium spinning the plate at 500 x g for 3 min at room temperature. The supernatant from the first wash can be removed by flicking the plate. Carefully remove the supernatant from the second wash using a multichannel pipette to make sure the entire volume of washing medium is removed. Do not disturb the pellet.</li>
		<li>Re-suspend the EtBr-labeled IEs in 30 &#956;L of antibody/plasma/serum solution prepared at the desired concentrations in parasite culture medium.</li>
		<li>Always include the following controls (Figure S2B): a control without IEs/THP-1 cells control (parasite culture medium); a control without any antibody or plasma/serum (un-opsonized control); a positive control using the commercially available rabbit anti-human erythrocyte antibody at 1:100 dilution (see Table of Materials); two negative plasma/serum controls at 1:5 dilution (a malaria-na&#239;ve pool and a pool from malaria-exposed males); and a positive serum control at 1:5 dilution (a pool from malaria-exposed women, who have previously been pregnant (preferably multigravidae)). 
		NOTE: A 1:100 dilution for the positive control seems to work consistently across different batches of purchased antibody (data not shown). However, it is recommended to rule out variations between batches by testing several dilutions every time a new batch of antibody is used.</li>
		<li>Incubate for 45 min in the dark at 37 &#176;C.</li>
	</ol>
	</li>
	<li>THP-1 cells preparation and phagocytosis
	<ol>
		<li>While the IEs are being opsonized (3.2.8), begin preparing the THP-1 cells.</li>
		<li>Remove the THP-1 cells from the culture flask, spin them down (500 x g for 5 min at room temperature), decant the supernatant and re-suspend the pellet in THP-1 cell culture medium.</li>
		<li>Spin again (500 x g for 5 min at room temperature), decant the supernatant and re-suspend the cell pellet in 1 mL of THP-1 cell culture medium.</li>
		<li>Determine cell count in the solution prepared above and add more medium to obtain a final concentration of 5 x 105 cells/mL.</li>
		<li>Remove the blocking solution from the remaining 96 well plate (phagocytosis plate) by flicking the plate and removing liquid excess over a piece of towel paper.</li>
		<li>Add 100 &#956;L per well of the THP-1 cell suspension prepared in 3.3.4 and put back in the cell culture incubator (Figure S2C).</li>
		<li>Once the antibody/plasma/serum incubation time has finished, add 170 &#181;L per well of parasite culture medium. Spin the plate at 500 x g for 3 min at room temperature and remove the supernatant by flicking the plate over an appropriate waste container.</li>
		<li>Wash the opsonized IEs two more times using 200 &#181;L per well of parasite culture medium, spinning the plate at 500 x g for 3 min at room temperature. The supernatant from the first wash can be removed by flicking the plate. Carefully remove the supernatant from the second wash, using a multichannel pipette to make sure the entire volume of washing medium is removed. Do not disturb the pellet.</li>
		<li>Finally re-suspend the opsonized IEs in 100 &#181;L of pre-warmed THP-1 cell culture medium. Transfer 50 &#181;L of opsonized IE suspension to each well in the phagocytosis plate. Since there is a total of 100 &#181;L of IEs, each antibody/serum dilution can be run in duplicates in the phagocytosis plate (Figure S2D) 
		NOTE: The amount of IEs and THP-1 cells used in the assay correspond to a 10:1 ratio.</li>
		<li>Incubate for 40 min in the dark at 37 &#176;C, 5% CO2. 
		NOTE: Do not allow the phagocytosis to proceed for more than 40 min.</li>
		<li>Stop the phagocytosis by centrifugation at 4 &#176;C (500 x g, 5 min) and discard the supernatant by flicking the plate. Add 150 &#181;L of room-temperature ammonium chloride lysing solution (Table of Materials) and mix by pipetting, incubate for exactly 3 min. 
		NOTE: This step will lyse the erythrocytes that have not been phagocytosed by the THP-1 cells.</li>
		<li>Stop the lysis by adding 100 &#181;L of ice-cold 2% FBS in PBS. Spin the plate at 4 &#176;C (500 x g for 3 min) and remove the supernatant by flicking the plate over an appropriate waste container.</li>
		<li>Wash three times using 200 &#181;L per well of ice-cold 2% FBS in PBS spinning the plate at 4&#176;C (500 x g for 3 min). After the final wash, re-suspend in 200 &#181;L of ice-cold 2% FBS in PBS and immediately analyze by flow cytometry. 
		NOTE: Previous publications have used cell fixation in 2% paraformaldehyde prior to flow cytometry31, but the results presented here were acquired immediately after assay completion. Postponing flow cytometry data acquisition by storing the plate at 4&#176;C is not recommended, since the percentage of EtBr+ THP-1 cells decays rapidly (Figure S3).</li>
	</ol>
	</li>
	<li>Flow cytometry acquisition and analysis 
	&#8203;NOTE: Any flow cytometer supporting 96 well plate format and having the appropriate lasers/filters to measure EtBr fluorescence can be used.
	<ol>
		<li>For acquisition, gate on THP-1 cells using a linear forward-scatter (FSC) vs. linear side-scatter (SSC) plot using the wells were no IEs were added (Figure 2A) and acquire 10,000 events on this gate.</li>
		<li>Measure fluorescence intensity for EtBr (FL3-Log) on the THP-1 gate using a histogram plot.</li>
		<li>For gating, first gate on THP-1 cells in a FSC vs. SSC density plot, using the wells were no IEs were added (Figure 2A). Then set up a positive gate in an FL3 (EtBr) histogram, using the THP-1 cells (no IEs added) and the un-opsonized control (Figure 2B).</li>
		<li>Copy these gates in all the other samples tested in the same plate and determine the percentage of EtBr-positive THP-1 cells (THP-1 cells that have phagocytized at least one IE). For each of the samples tested, phagocytosis can be reported as the absolute values (percentage of EtBr+ THP-1 cells) or as relative phagocytosis calculated as percentage using the positive control as maximum.</li>
	</ol>
	</li>
</ol>

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Part I. Learner's Guide.</a> World Health Organization. , (1991).</li><li>Tsuchiya, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+a+human+acute+monocytic+leukemia+cell+line+(THP-1).">Establishment and characterization of a human acute monocytic leukemia cell line (THP-1).</a> International Journal of Cancer. Journal International Du Cancer. 26 (2), 171-176 (1980).</li><li>Fleit, H. B., Kobasiuk, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+monocyte-like+cell+line+THP-1+expresses+FcyRl+and+Fc'yRIl.">The human monocyte-like cell line THP-1 expresses FcyRl and Fc'yRIl.</a> Journal of Leukocyte Biology. 49, 556-565 (1991).</li><li>Auwerx, J., Staels, B., Van Vaeck, F., Ceuppens, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+IgG+Fc+receptor+expression+induced+by+phorbol+12-myristate+13-acetate+treatment+of+THP-1+monocytic+leukemia+cells.">Changes in IgG Fc receptor expression induced by phorbol 12-myristate 13-acetate treatment of THP-1 monocytic leukemia cells.</a> Leukemia research. 16 (3), 317-327 (1992).</li><li>Ata&#237;de, R., 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=Using+an+improved+phagocytosis+assay+to+evaluate+the+effect+of+HIV+on+specific+antibodies+to+pregnancy-associated+malaria.">Using an improved phagocytosis assay to evaluate the effect of HIV on specific antibodies to pregnancy-associated malaria.</a> PloS One. 5 (5), 10807 (2010).</li></ol>

The authors have nothing to disclose.

The protocol presented here has been previously described and used12,15,17,31 to measure the capacity of antibodies targeting the surface of P. falciparum IEs to induce opsonization and phagocytosis by THP-1 cells. The results presented here focus on naturally acquired VAR2CSA-specific antibodies in the plasma/serum of women living in a P. falciparum endemic region. VAR2CSA is...

Malaria is a vector-borne disease caused in humans upon infection with five different species of the genus Plasmodium. The most prevalent species is P. falciparum, which is also responsible for the most morbidity and mortality1. Malaria clinical presentation varies from asymptomatic or benign infections to complicated/severe disease, the latter occurring mostly in children under the age of five years. Exposure to P. falciparum does not induce sterile immunity, but individuals living in endemic areas slowly develop immunity against the clinical disease. Protection is age/exposure dependent and immunity is normally acquired during the first 5-10 years of life2. Adult women are an important exception, as severe malaria can occur during pregnancy in a clinical presentation known as placental malaria (PM). PM is an important cause of abortion, stillbirth, premature delivery, low birth weight, fetal death, and maternal anemia. Resistance to PM develops over successive pregnancies3. Protection from PM is associated with the acquisition of antibodies against VAR2CSA-type PfEMP14,5, an infected erythrocyte (IE) surface antigen that binds to chondroitin sulphate A (CSA) enabling IE sequestration in the placenta. Antibodies mediate protection performing various functional activities (reviewed in6,7) including opsonization of IEs to induce phagocytosis. Early in vitro studies showed that antibodies can limit P. falciparum growth in the presence of monocytes via phagocytosis8,9. More recent studies have shown that higher levels of phagocytosis-inducing antibodies are associated with better pregnancy outcomes (in the context of HIV co-infection)10,11, indicating the relevance of this effector function in the naturally acquired immune response.
Here we present a protocol to measure this function of antibodies present in human plasma/serum, using in vitro cultured IEs expressing VAR2CSA together with the monocyte line THP-1. The assay has been previously used11,12,13,14,15,16,17,18 and is considered an improved and easier approach compared to earlier microscope-based protocols8, since it allows testing of a larger number of antibody samples in a single run using smaller volumes of antibody and avoiding tedious and biased microscopy counting. Even though the assay has been used by multiple laboratories and its execution is simple enough, it requires careful planning and preparation, therefore, a detailed protocol would allow its application by laboratories and researchers lacking previous experience. We use, as an example, late-stage-synchronized IEs expressing VAR2CSA opsonized with antibodies present in serum collected from women with naturally acquired PM-specific immunity. 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.

<table>
	<tbody>
		<tr>
			<td>96 well cell culture plates, round bottom with lid</td>
			<td>Corning</td>
			<td>3799</td>
			<td>Any similar plate can be used, make sure it is compatible with the flow cytometer instrument you intend to use</td>
		</tr>
		<tr>
			<td>AlbuMAX-II</td>
			<td>Gibco</td>
			<td>11021-037</td>
			<td></td>
		</tr>
		<tr>
			<td>AlbuMAX-II (5%)</td>
			<td>-</td>
			<td>-</td>
			<td>5% AlbuMAX-II (Gibco, 11021-037), 0.2g/L hypoxanthine (Sigma, H9377) in RPMI1640 (Sigma, R5886)</td>
		</tr>
		<tr>
			<td>Anti-Red Blood Cells antibody</td>
			<td>Abcam</td>
			<td>ab34858</td>
			<td>Prepare 2&#65533;&#65533;l aliquots and freeze a -20&#176;C. Use one aliquot per experiment.</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Sigma</td>
			<td>P8622</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads Protein G</td>
			<td>Invitrogen</td>
			<td>10003D</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium bromide solution</td>
			<td>Sigma</td>
			<td>E1510</td>
			<td>Prepare a stock solution at 0.1mg/mL in RPMI1640 (R5886). Store protected from light</td>
		</tr>
		<tr>
			<td>FC500 flow cytometer</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>Any flow cytometer supporting 96 well plate format and having the appropriate lasers/filters to measure EtBr fluorescence can be used.</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>10099-141</td>
			<td>Heat inactivate before use.</td>
		</tr>
		<tr>
			<td>FITC mouse anti-human CD16</td>
			<td>BD Biosciences</td>
			<td>555406 or 556618</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC mouse anti-human CD32</td>
			<td>BD Biosciences</td>
			<td>552883</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC mouse anti-human CD64</td>
			<td>BD Biosciences</td>
			<td>555527</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowLogic software</td>
			<td>Inivai technologies</td>
			<td></td>
			<td>Any flow cytometry analysis can be used, for example FlowJo or Winlist</td>
		</tr>
		<tr>
			<td>Gentamicin (10mg/mL)</td>
			<td>Sigma</td>
			<td>G1272</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypoxanthine</td>
			<td>Sigma</td>
			<td>H9377</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine (200mM)</td>
			<td>Sigma</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysing solution</td>
			<td>-</td>
			<td>-</td>
			<td>15mM NH4Cl, 10mM NaHCO3, 1mM EDTA</td>
		</tr>
		<tr>
			<td>MACS CS-column and accesories</td>
			<td>Miltenyi Biotec</td>
			<td>130-041-305</td>
			<td></td>
		</tr>
		<tr>
			<td>Parasite culture medium</td>
			<td>-</td>
			<td>-</td>
			<td>2mM L-glutamine (Sigma, G7513), 50&#181;g/mL Gentamicin (Sigma, G1272), 0.5% AlbuMAX-II (AlbuMAX-II 5%) in RPMI1640 (Sigma, R5886)</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (10000U and 10mg/mL)</td>
			<td>Sigma</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 medium</td>
			<td>Sigma</td>
			<td>R5886</td>
			<td></td>
		</tr>
		<tr>
			<td>THP-1 culture medium</td>
			<td>-</td>
			<td>-</td>
			<td>10%FBS (Gibco, 10099-141), 2mM L-glutamine (Sigma, G7513), 100U/mL Penicillin, 0.1mg/mL Streptomycin (Sigma, P0781) in RPMI1640 (Sigma, R5886)</td>
		</tr>
		<tr>
			<td>Vario MACS magnet</td>
			<td>Miltenyi Biotec</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Here we present in detail a protocol that has previously been described31 and used11,12,13,14,15,16,17,18 to measure the capacity of antibodies targeting the surface of P. falciparum IEs to induce opsonization and phagocytos...

Watch this Scientific Journal Video about Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay at JoVE.com

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).

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 ...

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6.3K Views.  University of Copenhagen. 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).

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

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 ...

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 ...

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.

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.

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

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 ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...