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>

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=World+Malaria+Report+-+2018.">World Malaria Report - 2018.</a> World Health Organization. , (2018).</li><li>Pierce, S. K., Miller, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=World+Malaria+Day+2009:+What+malaria+knows+about+the+immune+system+that+immunologists+still+do+not.">World Malaria Day 2009: What malaria knows about the immune system that immunologists still do not.</a> Journal of Immunology. 182 (9), 5171-5177 (2009).</li><li>Gamain, B., Smith, J. D., Viebig, N. K., Gysin, J., Scherf, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pregnancy-associated+malaria:+Parasite+binding,+natural+immunity+and+vaccine+development.">Pregnancy-associated malaria: Parasite binding, natural immunity and vaccine development.</a> International Journal for Parasitology. 37 (3-4), 273-283 (2007).</li><li>Staalsoe, T., 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=Variant+surface+antigen-specific+IgG+and+protection+against+clinical+consequences+of+pregnancy-associated+Plasmodium+falciparum+malaria.">Variant surface antigen-specific IgG and protection against clinical consequences of pregnancy-associated Plasmodium falciparum malaria.</a> Lancet. 363 (9405), 283-289 (2004).</li><li>Feng, G., 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=Antibodies+to+variant+surface+antigens+of+plasmodium+falciparum+-Infected+erythrocytes+are+associated+with+protection+from+treatment+failure+and+the+development+of+anemia+in+pregnancy.">Antibodies to variant surface antigens of plasmodium falciparum -Infected erythrocytes are associated with protection from treatment failure and the development of anemia in pregnancy.</a> The Journal of Infectious Diseases. 200 (2), 299-306 (2009).</li><li>Teo, A., Feng, G., Brown, G. V., Beeson, J. G., Rogerson, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Antibodies+and+Protection+against+Blood-stage+Malaria.">Functional Antibodies and Protection against Blood-stage Malaria.</a> Trends in Parasitology. 32 (11), 1-12 (2016).</li><li>Aitken, E. H., Mahanty, S., Rogerson, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+effector+functions+in+malaria+and+other+parasitic+diseases:+a+few+needles+and+many+haystacks.">Antibody effector functions in malaria and other parasitic diseases: a few needles and many haystacks.</a> Immunology &amp; Cell Biology. 98 (4), 264-275 (2020).</li><li>Celada, A., Cruchaud, A., Perrin, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opsonic+activity+of+human+immune+serum+on+in+vitro+phagocytosis+of+Plasmodium+falciparum+infected+red+blood+cells+by+monocytes.">Opsonic activity of human immune serum on in vitro phagocytosis of Plasmodium falciparum infected red blood cells by monocytes.</a> Clinical and Experimental Immunology. 47 (3), 635-644 (1982).</li><li>Celada, A., Cruchaud, A., Perrin, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+of+Plasmodium+falciparum-Parasitized+Erythrocytes+by+Human+Polymorphonuclear+Leukocytes.">Phagocytosis of Plasmodium falciparum-Parasitized Erythrocytes by Human Polymorphonuclear Leukocytes.</a> The Journal of Parasitology. 69 (1), 49-53 (1983).</li><li>Jaworowski, A., 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=Relationship+between+human+immunodeficiency+virus+type+1+coinfection,+anemia,+and+levels+and+function+of+antibodies+to+variant+surface+antigens+in+pregnancy-associated+malaria.">Relationship between human immunodeficiency virus type 1 coinfection, anemia, and levels and function of antibodies to variant surface antigens in pregnancy-associated malaria.</a> Clinical and Vaccine Immunology. 16 (3), 312-319 (2009).</li><li>Ata&#237;de, R., Mwapasa, V., Molyneux, M. E., Meshnick, S. R., Rogerson, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+that+induce+phagocytosis+of+malaria+infected+erythrocytes:+Effect+of+HIV+infection+and+correlation+with+clinical+outcomes.">Antibodies that induce phagocytosis of malaria infected erythrocytes: Effect of HIV infection and correlation with clinical outcomes.</a> PLoS One. 6 (7), 22491 (2011).</li><li>Ghumra, A., 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=Immunisation+with+recombinant+PfEMP1+domains+elicits+functional+rosette-inhibiting+and+phagocytosis-inducing+antibodies+to+Plasmodium+falciparum.">Immunisation with recombinant PfEMP1 domains elicits functional rosette-inhibiting and phagocytosis-inducing antibodies to Plasmodium falciparum.</a> PloS One. 6 (1), 0016414 (2011).</li><li>Ghumra, A., 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=Induction+of+strain-transcending+antibodies+against+Group+A+PfEMP1+surface+antigens+from+virulent+malaria+parasites.">Induction of strain-transcending antibodies against Group A PfEMP1 surface antigens from virulent malaria parasites.</a> PLoS Pathogens. 8 (4), 1002665 (2012).</li><li>Chan, J. A., 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=Targets+of+antibodies+against+erythrocytes+in+malaria+immunity.">Targets of antibodies against erythrocytes in malaria immunity.</a> J Clin Invest. 122 (9), 3227-3238 (2012).</li><li>Quintana, M. P., Angeletti, D., Moll, K., Chen, Q., Wahlgren, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+inducing+antibodies+to+Plasmodium+falciparum+upon+immunization+with+a+recombinant+PfEMP1+NTS+DBL1&#945;+domain.">Phagocytosis inducing antibodies to Plasmodium falciparum upon immunization with a recombinant PfEMP1 NTS DBL1&#945; domain.</a> Malaria Journal. 1, 1-9 (2016).</li><li>Chan, J. A., 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=A+single+point+in+protein+trafficking+by+Plasmodium+falciparum+determines+the+expression+of+major+antigens+on+the+surface+of+infected+erythrocytes+targeted+by+human+antibodies.">A single point in protein trafficking by Plasmodium falciparum determines the expression of major antigens on the surface of infected erythrocytes targeted by human antibodies.</a> Cellular and Molecular Life Sciences. 73, 4141-4158 (2016).</li><li>Quintana, M. P., 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=Antibodies+in+children+with+malaria+to+PfEMP1,+RIFIN+and+SURFIN+expressed+at+the+Plasmodium+falciparum+parasitized+red+blood+cell+surface.">Antibodies in children with malaria to PfEMP1, RIFIN and SURFIN expressed at the Plasmodium falciparum parasitized red blood cell surface.</a> Scientific Reports. 8 (1), 3262 (2018).</li><li>Hommel, M., 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=Evaluating+antibody+functional+activity+and+strain-specificity+of+vaccine+candidates+for+malaria+in+pregnancy+using+in+vitro+phagocytosis+assays.">Evaluating antibody functional activity and strain-specificity of vaccine candidates for malaria in pregnancy using in vitro phagocytosis assays.</a> Parasites &amp; Vectors. 11 (69), 1-7 (2018).</li><li>Ampomah, P., Stevenson, L., Ofori, M. F., Barfod, L., Hviid, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-cell+responses+to+pregnancy-restricted+and+-unrestricted+Plasmodium+falciparum+erythrocyte+membrane+protein+1+antigens+in+Ghanaian+women+naturally+exposed+to+malaria+parasites.">B-cell responses to pregnancy-restricted and -unrestricted Plasmodium falciparum erythrocyte membrane protein 1 antigens in Ghanaian women naturally exposed to malaria parasites.</a> Infection and Immunity. 82 (5), 1860-1871 (2014).</li><li>Cranmer, S. L., Magowan, C., Liang, J., Coppel, R. L., Cooke, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+alternative+to+serum+for+cultivation+of+Plasmodium+falciparum+in+vitro.">An alternative to serum for cultivation of Plasmodium falciparum in vitro.</a> Transactions of the Royal Society of Tropical Medicine and Hygiene. 91 (3), 363-365 (1997).</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> Journal of Parasitology. 65 (3), 418-420 (1979).</li><li>Barfod, L., 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=Human+pregnancy-associated+malaria-specific+B+cells+target+polymorphic,+conformational+epitopes+in+VAR2CSA.">Human pregnancy-associated malaria-specific B cells target polymorphic, conformational epitopes in VAR2CSA.</a> Molecular Microbiology. 63 (2), 335-347 (2007).</li><li>Staalsoe, T., 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=In+vitro+selection+of+Plasmodium+falciparum+3D7+for+expression+of+variant+surface+antigens+associated+with+severe+malaria+in+African+children.">In vitro selection of Plasmodium falciparum 3D7 for expression of variant surface antigens associated with severe malaria in African children.</a> Parasite Immunology. 25 (8-9), 421-427 (2003).</li><li>Chan, S., 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=Regulation+of+PfEMP1-VAR2CSA+translation+by+a+Plasmodium+translation-enhancing+factor.">Regulation of PfEMP1-VAR2CSA translation by a Plasmodium translation-enhancing factor.</a> Nature Microbiology. 2, 17068 (2017).</li><li>Barfod, L., 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=Evasion+of+immunity+to+Plasmodium+falciparum+malaria+by+IgM+masking+of+protective+IgG+epitopes+in+infected+erythrocyte+surface-exposed+PfEMP1.">Evasion of immunity to Plasmodium falciparum malaria by IgM masking of protective IgG epitopes in infected erythrocyte surface-exposed PfEMP1.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (30), 12485-12490 (2011).</li><li>Moll, K., Kaneko, A., Scherf, A., Wahlgren, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Malaria Research. , (2013).</li><li>WHO. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+malaria+microscopy.+Part+I.+Learner's+Guide.">Basic malaria microscopy. 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&amp;#956;l aliquots and freeze a -20&amp;deg;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&amp;micro;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 ...

measuring-naturally-acquired-phagocytosis-inducing-antibodies-to

Research

JoVE Journal

Immunology and Infection

6.4K Views.  University of Copenhagen. Dit protocol maakt het mogelijk om het vermogen van antilichamen te meten om opsoniseren en induceren de fagocytose van plasmodium falciparum geïnfecteerde erytrocyten. Antilichamen die aanwezig zijn in het serum van personen die van nature zijn blootgesteld aan parasietinfectie, evenals antilichamen die worden veroorzaakt door immunisatie met parasietantigenen, kunnen worden getest. Het aantonen van de procedure met mij zal Maiken Visti, een technicus van mijn laboratorium.Om een THP...

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

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 ...

Detection and Quantification of Plasmodium falciparum in Aqueous Red Blood Cells by Attenuated Total Reflection Infrared Spectroscopy and Multivariate Data Analysis

We demonstrate a method of quantification and detection of parasites in aqueous red blood cells (RBCs) by using a simple benchtop Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectrometer in conjunction with Multivariate Data Analysis (MVDA). 3D7 P. falciparum were cultured to 10% parasitemia ring stage parasites and used to spike fresh donor isolated RBCs to create a dilution series between 0&#8211;1%. 10 &#181;L of each sample were placed onto the center of the ATR diamond window to acquire the spectrum. The sample data was treated to improve the signal to noise ratio and to remove the contribution of water, and then the second derivative was applied to resolve spectral features. The data were then analyzed using two types of MVDA: first Principal Component Analysis (PCA) to determine any outliers and then Partial Least Squares Regression (PLS-R) to build the quantification model.

Detection and Quantification of Plasmodium falciparum in Aqueous Red Blood Cells by Attenuated Total Reflection Infrared Spectroscopy and Multivariate Data Analysis

Here, we present a protocol for the detection and quantification of Plasmodium falciparum in infected aqueous red blood cells using an attenuated total reflection infrared spectrometer and multivariate data analysis.

We demonstrate a method of quantification and detection of parasites in aqueous red blood cells (RBCs) by using a simple benchtop Attenuated Total ...

Chemistry

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 ...

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

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

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

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

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

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Biology

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This parasitic disease requires a protozoan parasite, an Anopheles mosquito vector, and a vertebrate host. The Anopheles genus comprises more than 500 species, of which 60 are known as vectors of the parasite. The Plasmodium parasite genus consists of 250 species, and 48 of these are involved in disease transmission. Furthermore, the Plasmodium falciparum parasite has contributed toward an estimated 99.7% of malaria cases in sub-Saharan Africa in recent years. 
Gametocytes form part of the sexual stage of the parasite and are ingested by the female mosquito upon feeding on an infected human host. Further development of the parasite within the mosquito is enhanced by favorable environmental conditions in the midgut of the mosquito. Here, the fusion of the female and male gametes takes place, and the motile ookinetes originate. The ookinetes enter the midgut epithelium of the mosquito, and mature ookinetes form oocysts, which, in turn, produce motile sporozoites. These sporozoites migrate to the mosquito's salivary glands and are injected as a mosquito takes a blood meal. 
For drug discovery purposes, mosquitoes were artificially infected with gametocyte-infected blood in the standard membrane feeding assay (SMFA). To detect infection within the mosquito and/or to assess the efficacy of antimalarial compounds, the midguts of the female mosquitoes were removed post infection and were stained with mercurochrome. This method was used to enhance the visual detection of oocysts under the microscope for the accurate determination of oocyst prevalence and intensity.

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

The standard membrane feeding assay (SMFA) is regarded as the gold standard for the assessment and identification of potential antimalarial compounds. This artificial feeding system is used to infect mosquitoes to further evaluate the effects of such compounds on the intensity and prevalence of the Plasmodium falciparum parasite.

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This ...

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

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

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

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

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

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female Anopheles mosquitoes are transmitted to humans via mosquito bites during blood feeding. The presence of sporozoites in the mosquito salivary glands thus defines mosquito infectiousness. To determine whether an Anopheles mosquito carries Plasmodium sporozoites, the enzyme-linked immunosorbent assay (ELISA) method has been the standard tool to detect the Plasmodium circumsporozoite protein (CSP), the major surface protein of the sporozoites. In this method, the head along with the thorax of each mosquito is separated from the abdomen, homogenized, and subjected to a sandwich ELISA to detect the presence of CSP specific to Plasmodium falciparum and each of the two subtypes, VK210 and VK247, of Plasmodium vivax.This method has been used to study malaria transmission, including the seasonal dynamics of mosquito infection and the species of the major malaria vectors in the study sites.

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

This protocol describes a sandwich enzyme-linked immunosorbent assay to detect salivary gland sporozoites in mosquitoes. Using easily available monoclonal antibodies, the method enables cost-effective, high-throughput detection of mosquitoes carrying Plasmodium falciparum or Plasmodium vivax. The method is suitable for malaria transmission research, including vector surveys.

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

Samenvatting

Meer video's verkennen