This study was financially supported in part by the Novartis foundation for medical- and biological research (to PYM), the Gottfried and Julia Bangerter-Rhyner-Stiftung (to MW and PYM), and the research pool of the University of Fribourg (to PYM). Additional grants include the Swiss Government Excellence Scholarships for Foreign Scholars (to KAB and SM). We thank Isabelle Fellay and Solange Kharoubi Hess for technical support.

Human RBCs were obtained from the blood of healthy donors, in accordance with the guidelines of Swissethics (swissethics.ch).
NOTE: P. falciparum parasite cultures (3D7) and EV production were previously described in Mbagwu, et al.11 Because P. falciparum is a human pathogen, consult the local regulations for handling. The cultures should be kept sterile the entire time.
1. Fluorescence Labeling of EVs
NOTE: The following procedure takes advantage of the labeling technology to stably incorporate a green fluorescent dye (PKH67) with large aliphatic tails in the lipid region of the EV membrane. The reaction is performed in a 200 &#181;L final staining volume containing a 20 &#181;M final concentration of PKH67. Perform all steps at ambient temperature (20-25 &#176;C)
<ol>
	<li>Place 100 &#181;g of EVs in 1 mL of PBS in a microcentrifuge tube.</li>
	<li>Centrifuge the EVs in a microcentrifuge tube at maximum speed (20,000 x g) for 7 min to pellet. 
	NOTE: Since hemozoin is present in EVs, a red-dark brownish pellet should be observed. If the supernatant is reddish, then the EVs have lysed.</li>
	<li>After centrifugation, aspirate the supernatant carefully, in order to avoid removing any EVs. 
	NOTE: Minimize the amount of remaining buffer present before resuspending EVs in diluent C in order to increase reproducibility and efficiency of the staining.</li>
	<li>Resuspend the EV pellet in 100 &#181;L of diluent C (2X EV Suspension) and gently pipet to insure complete dispersion.</li>
	<li>Prepare a new microcentrifuge tube and add 4 &#181;L of the PKH67 ethanolic dye solution to 100 &#181;L of diluent C. Vortex well to mix. Prepare this 2x dye solution (40 &#181;M) immediately prior to staining. 
	NOTE: To avoid heterogeneous staining, do not add the PKH67 ethanolic dye solution directly to the EV pellet.</li>
	<li>Rapidly, combine the 100 &#181;L of 2X EV Suspension (step 1.4) with the 100 &#181;L of 2X dye solution (step 1.5). Then, mix the sample by pipetting up and down 10 times.</li>
	<li>Incubate the EV/Dye suspension from step 1.6 for 5 min protected from light and mix by vortexing 3-4 times during the 5 min incubation. 
	NOTE: Incubating the EV/Dye suspension for longer times provides no advantage, because the staining is so rapid.</li>
	<li>Stop the staining reaction by adding an equal volume (200 &#181;L) of serum and incubate for 1 min to allow binding of excess dye. 
	NOTE: Before stopping the staining, deplete the serum from EVs by centrifugation at 20,000 x g for 20 min.</li>
	<li>Wash 3 times with PBS and spin down at 20,000 x g for 5 min. Resuspend the EVs in 100 &#181;L of PBS to reach a final concentration of 1 &#181;g/&#181;L.</li>
</ol>
2. Visualize Uptake of EVs by Confocal Microscopy
NOTE: The following protocol describes the tracking of EV internalization by endothelial cells grown on glass coverslips. The endothelial cells are semi-immortalized human Bone Marrow Endothelial cells as described in12.
<ol>
	<li>Working in a sterile environment, coat coverslips with an excess of 0.01% poly-L-lysine for 10 min at room temperature (RT).</li>
	<li>Aspirate the poly-L-lysine solution and allow the coverslips to dry completely.</li>
	<li>Perform a viable endothelial cell count using Trypan blue exclusion and a hemocytometer.</li>
	<li>Transfer 1 x 105 endothelial cells in 1 mL of complete Endothelial Cell Growth Medium containing the Supplement Mix to the poly-L-lysine-coated coverslips and let the cells grow to a monolayer. 
	NOTE: The medium contains growth factors that allow cells to form a monolayer and to form tight junctions.</li>
	<li>Once the cells form a monolayer, carefully aspirate the medium and add 1 mL of fresh medium to wash the cells. Repeat the wash once more. Add 50 &#181;g of PKH67-labeled EVs and incubate for 4 h. 
	NOTE: To find the optimal time-point for uptake, a time-course can be performed.</li>
	<li>After 4 h, aspirate the medium, and wash the cells 3 times with PBS to remove excess and unbound EVs. Fix the cells with 3% paraformaldehyde solution in PBS for 15 min. 
	NOTE: Methanol can disrupt actin during the fixation process; therefore, it is best to avoid any methanol containing fixatives or other solvent-based fixatives. It is recommended to use methanol-free formaldehyde as a fixative.</li>
	<li>Wash the cells 3 times with PBS. Add carefully 250 &#181;L of 0.1% Triton X-100 in PBS to permeabilize the cells. Incubate at RT for 5 min.</li>
	<li>Wash 3 times with PBS. Add 400 &#181;L of blocking buffer (5% BSA in PBS) for 30 min at RT to block non-specific binding.</li>
	<li>Wash the cells once with PBS. Prepare the staining solution by diluting 5 &#181;L of the phalloidin stock solution in 200 &#181;L PBS/1% BSA per each cover slip. Pipet 200 &#181;L of the staining solution on the coverslip and incubate for 20 min at RT.</li>
	<li>Wash 3 times with PBS. Counterstain the DNA for 30 s with Hoechst 33342 at a final concentration of 2 &#181;g/mL in 200 &#181;L of PBS.</li>
	<li>Wash 2x the coverslip by adding 400 &#181;L of PBS. Carefully grab the coverslip with forceps and invert it on the top of a drop of antifade mounting media on a glass slide. Remove gently the excess media with a tissue and seal each side with nail polish.</li>
	<li>Store the slides protected from the light at 4 &#176;C.</li>
	<li>Visualize the cells using a confocal microscope at laser excitations 405, 488, and 543 nm and a 63X, 1.3 NA oil objective with fluorescent emissions at 450-470 nm (blue), 505-530 nm (green), and 620 nm. 
	NOTE: Typical F-actin staining results are shown in Figure 1.</li>
</ol>
3. Endothelial Cell Permeability
NOTE: Endothelial cell permeability is assessed by measuring the transfer of rhodamine B isothiocyanate-dextran (average MW 70,000) across the endothelial cell monolayer. Dextran provides an excellent tool to study vascular permeability.
<ol>
	<li>Count viable endothelial cells using 0.4 % Trypan blue exclusion and a hemocytometer.</li>
	<li>Plate the endothelial cells at a density of 0.6 x 105 cells to confluence in 24-well tissue culture inserts (0.4 &#181;m pore size) and leave undisturbed for at least 5 days to form differentiated monolayers. Refresh the medium every second day.</li>
	<li>Once the cells reach a monolayer, load the EVs (50 &#181;g in 1 mL) on the upper chamber. Add the rhodamine-dextran to the top well at a final concentration of 20 mg/mL. Incubate the cells at 37 &#176;C with 5% CO2.</li>
	<li>Monitor the transfer of the fluorescent dextran to the bottom well by measuring fluorescence emission from 40 &#181;L medium aliquots. Set up the multi-label microplate reader at 544 nm excitation and 590 nm emission for measurements. 
	NOTE: The amount of diffused dextran can be quantified using calibration curves established with the stock solution. In addition, kinetic experiments can be performed by removing small samples of the outer chamber medium during a time course (Figure 2). Even without any treatment, the dextran will diffuse through the cell monolayer.</li>
</ol>
4. Determine Puromycin Sensitivity
NOTE: Before transducing the cell lines with lentivirus, it is important to determine the kill curve of a selected drug for that particular cell line. To determine the minimum amount of drug concentration necessary to kill all of the cells, perform a dose response experiment using incremental doses of the selected drug. Because each mammalian cell line has a different sensitivity, before experimentation, the optimal concentration of the antibiotic should be determined by developing the kill curve titration as detailed below.
<ol>
	<li>Count the cells using the hemocytometer. Resuspend the endothelial cells at a concentration of 1 x 105 cells/mL in complete Endothelial Cell Growth Medium containing the Supplement Mix.</li>
	<li>Plate 1 x 104 cells into a 96-well plate in a final volume of 100 &#181;L of the Endothelial Cell Growth Medium.</li>
	<li>Add 100 &#181;L of medium containing the antibiotics to obtain a concentration of 0.1-10 &#181;g/mL (see Figure 3).</li>
	<li>Examine the cell viability every 2 days under a light microscope using a 20X objective. Culture the cells for 10-14 days and replace the media containing puromycin every 2-3 days depending on cell growth.</li>
	<li>Add 10 &#181;L/well MTS Reagent into each well, mix, and incubate for 4 h at 37 &#176;C in standard culture conditions.</li>
	<li>Shake the plate briefly on a shaker and measure absorbance of treated and untreated cells using a plate reader at 490 nm (Figure 3).</li>
</ol>
5. Transduction of Endothelial Cells with Lentiviral Vector
<ol>
	<li>Plate 1 x 105 endothelial cells in 1 mL of complete medium the day before transduction. Perform transduction when the cells have reached 50-80% confluency.</li>
	<li>Thaw the lentivirus on ice, and make aliquots of the viral stocks to avoid freeze-thaw cycles. Spin down the material at 200 x g for 30 s, in tubes before opening to avoid spill over.</li>
	<li>Add hexadimethrine bromide to the cells at a final concentration of 8 &#181;g/mL. 
	NOTE: Hexadimethrine bromide helps the transduction efficiency, however in some cases it might be toxic for the cells. Therefore, the optimal concentration should be determined by performing a killing curve before the transduction.</li>
	<li>Swirl the plate gently to mix. Add the appropriate quantity of viral particles (between 0.5-2 x 106 particles) at a suitable multiplicity of infection (MOI) and swirl the plate gently for 30 s to mix.</li>
	<li>Centrifuge the cell-viral particle mix in the centrifuge at RT for 45 min at 300 x g to increase the transduction efficiency. Incubate the cell-viral particle mixture at 37 &#176;C overnight.</li>
	<li>Remove carefully the viral particle-containing medium and discard appropriately. Replace it with fresh, pre-warmed complete culture medium.</li>
	<li>Start the selection with puromycin on the second day: remove the medium and replace it with fresh medium containing the correct concentration of puromycin as determined previously in section 4 (Figure 3).</li>
	<li>Harvest cells after puromycin selection and isolate RNA using an RNA isolating kit following the manufacturer&#39;s instructions.</li>
	<li>Perform cDNA synthesis with the selected miRNAs using a reverse transcription kit (see the Table of Materials).</li>
	<li>Set up the qPCR reaction according to the cited protocol13.</li>
</ol>

<ol>
	<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=.">.</a> WHO Malaria Report 2015. , (2015).</li><li>Miller, L. H., Baruch, D. I., Marsh, K., Doumbo, O. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathogenic+basis+of+malaria.">The pathogenic basis of malaria.</a> Nature. 415 (6872), 673-679 (2002).</li><li>Mantel, P. Y., Marti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+extracellular+vesicles+in+Plasmodium+and+other+protozoan+parasites.">The role of extracellular vesicles in Plasmodium and other protozoan parasites.</a> Cell Microbiol. 16 (3), 344-354 (2014).</li><li>Couper, K. 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Y., 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=Malaria-infected+erythrocyte-derived+microvesicles+mediate+cellular+communication+within+the+parasite+population+and+with+the+host+immune+system.">Malaria-infected erythrocyte-derived microvesicles mediate cellular communication within the parasite population and with the host immune system.</a> Cell Host Microbe. 13 (5), 521-534 (2013).</li><li>Regev-Rudzki, N., 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=Cell-cell+communication+between+malaria-infected+red+blood+cells+via+exosome-like+vesicles.">Cell-cell communication between malaria-infected red blood cells via exosome-like vesicles.</a> Cell. 153 (5), 1120-1133 (2013).</li><li>Szempruch, A. J., 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=Extracellular+Vesicles+from+Trypanosoma+brucei+Mediate+Virulence+Factor+Transfer+and+Cause+Host+Anemia.">Extracellular Vesicles from Trypanosoma brucei Mediate Virulence Factor Transfer and Cause Host Anemia.</a> Cell. 164 (1-2), 246-257 (2016).</li><li>Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+genomics,+biogenesis,+mechanism,+and+function.">MicroRNAs: genomics, biogenesis, mechanism, and function.</a> Cell. 116 (2), 281-297 (2004).</li><li>Mantel, P. Y., 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=Infected+erythrocyte-derived+extracellular+vesicles+alter+vascular+function+via+regulatory+Ago2-miRNA+complexes+in+malaria.">Infected erythrocyte-derived extracellular vesicles alter vascular function via regulatory Ago2-miRNA complexes in malaria.</a> Nat Commun. 7, 12727 (2016).</li><li>Mbagwu, S., Walch, M., Filgueira, L., Mantel, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+Characterization+of+Extracellular+Vesicles+in+Malaria.">Production and Characterization of Extracellular Vesicles in Malaria.</a> Methods Mol Biol. 1660, 377-388 (2017).</li><li>Schweitzer, K. 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=Characterization+of+a+newly+established+human+bone+marrow+endothelial+cell+line:+distinct+adhesive+properties+for+hematopoietic+progenitors+compared+with+human+umbilical+vein+endothelial+cells.">Characterization of a newly established human bone marrow endothelial cell line: distinct adhesive properties for hematopoietic progenitors compared with human umbilical vein endothelial cells.</a> Lab Invest. 76 (1), 25-36 (1997).</li><li>Wong, W., Farr, R., Joglekar, M., Januszewski, A., Hardikar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probe-based+Real-time+PCR+Approaches+for+Quantitative+Measurement+of+microRNAs.">Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs.</a> J Vis Exp. (98), (2015).</li><li>Mulcahy, L. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis,+clinical+features,+and+neurological+outcome+of+cerebral+malaria.">Pathogenesis, clinical features, and neurological outcome of cerebral malaria.</a> Lancet Neurol. 4 (12), 827-840 (2005).</li><li>Jambou, 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=Plasmodium+falciparum+adhesion+on+human+brain+microvascular+endothelial+cells+involves+transmigration-like+cup+formation+and+induces+opening+of+intercellular+junctions.">Plasmodium falciparum adhesion on human brain microvascular endothelial cells involves transmigration-like cup formation and induces opening of intercellular junctions.</a> PLoS Pathog. 6 (7), e1001021 (2010).</li><li>Zhou, W., 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=Cancer-secreted+miR-105+destroys+vascular+endothelial+barriers+to+promote+metastasis.">Cancer-secreted miR-105 destroys vascular endothelial barriers to promote metastasis.</a> Cancer Cell. 25 (4), 501-515 (2014).</li></ol>

The authors have nothing to disclose.

Several parasites, including Toxoplasma, Trypanosoma, Leishmania, and Trichomonas trigger the release of EVs by the infected host cell. Depending on the pathogens, the released EVs can modulate the host immune response or mediate cellular communication between the parasites6. Yet, there is little evidence suggesting how these small vesicles contribute to malaria disease. Here, we have described several ways to investigate the function of EVs during Plasmodium infection. For instance, the uptake of EVs by recipient cells in vivo can be efficiently monitored by labeling them with the PKH67 fluorescent dye. Several controls have to be performed in order to guarantee the specificity of the labeling. It is important to note that once mixed with the diluent C, the PKH67 dye tends to form aggregates, and it is therefore necessary to include a control with the dye alone without EVs to determine the non-specific staining. Confocal microscopy is the method of choice to differentiate between vesicles bound to the surface and truly internalized by the recipient cells. In addition, it provides an excellent tool to track and monitor the number of vesicles taken up by the cells. However, it remains unclear how EVs are processed in the recipient cells14; therefore, performing a time-course using time-points of 2, 4, and 8 h, may be very useful in order to determine the optimal conditions for uptake. The addition of subcellular compartment markers can be used to track the EVs inside the cells by confocal microscopy. EV uptake has been shown to occur via membrane fusion or endocytosis15. The fusion can be monitored by labeling EVs with octadecyl rhodamine B (R18). The fusion of EVs with the cellular membranes results in the dilution of R18, which leads to an increase in fluorescence16. Uptake of EVs by endocytosis can be studied by using specific inhibitors of actin, microtubule, and dynamin10.
To study the physiology of EVs, it is possible to label the iRBCs directly with the PKH dyes and co-culture the iRBCs with endothelial cells. The problem here is that the cells require different media and gas compositions and the amount of EV transfer might be low. Furthermore, vesicular proteins fused with a fluorescent tag can be expressed in the donor cells in vitro and in vivo. However, so far it has not been tested with iRBCs
Infected RBCs are cytoadherent to the microvasculature of the brain, in a process thought to be responsible to cause cerebral malaria17. Therefore, the transfer of parasite material via EVs is likely to influence endothelial cells and hence vascular function18. The in vitro assay described here, provides an easy way to investigate some of the basic properties of endothelial cells, as for example its permeability19. Additional test such as TEER or immunostaining with ZO-1 can provide more information about the tight junction integrity10.
Furthermore, the in vitro model turns out to be a powerful tool to study the molecular mechanisms behind the cellular communication between parasites and host cells. Besides uptake, this setup allows the investigation of molecular mechanisms involved in this process. For example, we have monitored the transfer of miR451a to the acceptor cells by Real-Time PCR and fluorescent in situ hybridization. Additionally, we describe how to generate cell lines overexpressing a particular miRNA, to directly address the role of these small RNA in vascular function10. However, several controls should be performed including testing non-transduced cells, since the transduction itself might affect the cellular function. Furthermore, miRNA inhibitors can be used to neutralize the effect of the miRNAs.

According to the World Health Organization, there were 212 million new cases of malaria worldwide in 2015 and approximately 429,000 people died, mainly children under five years of age1. The mechanisms leading to severe disease, which is often associated with vascular dysfunction, remain ill-defined2. Plasmodium-iRBCs secrete small bi-lipid membrane spheres known as extracellular vesicles (EVs). It is known that these EVs are potentially relevant to the infection process and to the host immune response to infection; however, little is known about the exact function of these small vesicles during malaria infection3. It is possible that they play two important roles: on one hand, they might contribute to the pathogenesis by activating macrophages4,5; and on the other hand, they might mediate cellular communication between parasites and between parasites and host6,7. In fact, parasites can transfer proteins or nucleic acids between each other via EVs. For example, Trypanosoma brucei rhodesiense EVs can transfer virulence factor Serum Resistance-Associated (SRA), and can target both other T. brucei and host erythrocytes8. Furthermore, P. falciparum-iRBCs communicate between each other by transferring nucleic acids within EVs. This allows the parasites to optimize and synchronize its growth. In fact, EVs might be the major regulator of gametocyte conversion, and therefore contribute to the regulation of the transmission stage7.
Not only do EVs regulate the parasites, they also mediate parasite-host interactions. We recently discovered that EVs from iRBCs contain host-derived microRNAs (miRNAs; small RNA species in the range of 21-25 nucleotides9) that were taken up by human endothelial cells. The miRNAs in the EVs form a stable complex with Ago2 (a member of the RNA-induced silencing complex), which once delivered to the recipient cells, is capable of specifically silencing gene expression and affecting the barrier properties of the cells10. Standard protocols have been developed to investigate the function of EVs. Here, we describe first a protocol that allows the fluorescent labeling of EVs to investigate their uptake by recipient cells. In addition, by using a confocal microscope, it is possible to track the EV&#39;s fate inside the cell. Several fluorescent dyes can be used to track EVs. The amine-reactive dye, 5-(and-6)-Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) and Calcein-AM become fluorescent once inside the vesicles. We prefer to use the amphiphilic label, PKH, because it gives a brighter and more uniform signal. This approach provides important information to understand the interactions between EVs and recipient cells. While in some cases EVs bind to the surface of the cells, some vesicles are rapidly taken up. Upon uptake, EVs deliver their cargoes to the cells, in which they exert their regulatory functions.
Here, we describe a protocol to measure the barrier function of the endothelial cells in vitro by quantifying the transfer of a fluorescent dextran through a cellular monolayer. More sensitive tracers can be used such as radiolabeled markers. However, they require special safety precautions for use. Other assays exist to measure the in vitro barrier function such as transendothelial electrical resistance (TEER), which measures tight junction integrity. Finally visualizing ZO-1, a tight junction protein, by immunofluorescence allows assessment of tight junction integrity as well10. Since EVs are complex and heterogeneous entities containing several cargoes with potential regulatory characteristic, it is useful to overexpress a specific RNA to study its effect on the recipient cell. Therefore, we also define a protocol that aims to generate stable cell lines expressing the miRNA of interest10.

<table>
	<tbody>
		<tr>
			<td>PKH67 Green Fluorescent Cell Linker Mini Kit</td>
			<td>Sigma-Aldrich</td>
			<td>MINI67-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Diluent C</td>
			<td>Sigma-Aldrich</td>
			<td>G8278</td>
			<td></td>
		</tr>
		<tr>
			<td>poly-L-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P8920</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>ThermoFisher - Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin CF594</td>
			<td>Biotium</td>
			<td>#00045</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>ThermoFisher</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>ThermoFisher</td>
			<td>P36934</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhodamine B isothiocyanate&#8211;Dextran</td>
			<td>ThermoFisher</td>
			<td>R9379-250MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Insert with PET membrane transparent Falcon&#160;for plate 24 wells</td>
			<td>Falcon</td>
			<td>353095</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth&#160;Medium MV</td>
			<td>Promocell</td>
			<td>C-22020</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin dihydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>P9620-10ML</td>
			<td></td>
		</tr>
		<tr>
			<td>MTS Cell Proliferation Colorimetric Assay Kit</td>
			<td>Biovision</td>
			<td>K300-500</td>
			<td></td>
		</tr>
		<tr>
			<td>hexadimethrine bromide</td>
			<td>Sigma-Aldrich</td>
			<td>107689-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>MISSION&#160;Lenti microRNA, Human hsa-miR-451a</td>
			<td>Sigma-Aldrich</td>
			<td>HLMIR0583</td>
			<td></td>
		</tr>
		<tr>
			<td>MISSION Lenti microRNA, ath-miR416, Negative Control 1 Transduction Particles</td>
			<td>Sigma-Aldrich</td>
			<td>NCLMIR001</td>
			<td></td>
		</tr>
		<tr>
			<td>MISSION&#160;Lenti microRNA, Human</td>
			<td>Sigma-Aldrich</td>
			<td>NCLMIR0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica TCS SP5</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>miRNeasy mini Kit</td>
			<td>Qiagen</td>
			<td>217004</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan MicroRNA Reverse Transcription Kit 1000 reactions</td>
			<td>ThermoFisher</td>
			<td>4366597</td>
			<td></td>
		</tr>
		<tr>
			<td>hsa-mir-451a&#160;RT/750 PCR rxns</td>
			<td>ThermoFisher</td>
			<td>001141</td>
			<td></td>
		</tr>
		<tr>
			<td>U6 snRNA</td>
			<td>ThermoFisher</td>
			<td>001973</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Universal Master Mix II, with UNG</td>
			<td>ThermoFisher</td>
			<td>4440038</td>
			<td></td>
		</tr>
		<tr>
			<td>StepOnePlus Real-Time PCR System</td>
			<td>ThermoFisher</td>
			<td>4376600</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of extracellular vesicle function during malaria infection

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible for most malaria-related deaths globally. Several factors including parasite sequestration in tissues, vascular dysfunction, and inflammatory responses influence the evolution of the disease in malaria-infected people. P. falciparum-infected red blood cells (iRBCs) release small extracellular vesicles (EVs) containing different kinds of cargo molecules that mediate pathogenesis and cellular communication between parasites and host. EVs are efficiently taken up by cells in which they modulate their function. Here we discuss strategies to address the role of EVs in parasite-host interactions. First, we describe a straightforward method for labeling and tracking EV internalization by endothelial cells, using a green cell linker dye. Second, we report a simple way to measure permeability across an endothelial cell monolayer by using a fluorescently labeled dextran. Finally, we show how to investigate the role of small non-coding RNA molecules in endothelial cell function.

Here, we describe protocols to investigate the interactions of EVs with host cells. The uptake of fluorescently labeled EVs is monitored by confocal microscopy (Figure 1). Endothelial cells efficiently take up EVs, however the incubation time with EVs can be optimized to track the uptake. For a better localization of EVs inside the cells, stain actin with phalloidin. Next, we use a filter membrane on top of which a monolayer of endothelial cells grows. Rhodamine B isothiocyanate-dextran is applied on the top chamber of the filter membrane and then the permeability of the endothelial monolayer is measured by monitoring the transfer of the fluorescent dextran into the bottom chamber (Figure 2A). Typically, the permeability of the endothelial cells is affected upon incubation with an increasing amount of EVs (Figure 2B). It is important to grow the cells for several days to make sure they will form a monolayer, otherwise the dye will quickly diffuse through the wells. It is important to note that even without treatment, the dextran will diffuse slowly through the cell monolayer.
MicroRNAs are key components of EVs and after transfer to the recipient cells, they regulate gene expression. In order to investigate specifically the role of miRNAs, it is very useful to overexpress them in the recipient cell line. Here we describe how to transduce miRNAs into endothelial cells. The first step consists of determining the concentration of puromycin that kills endothelial cells in order to select stably transduced cells (Figure 4A). Finally, the level of expression of the miRNAs can be controlled and validated by qPCR (Figure 4B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57067/57067fig1.jpg" /> 
Figure 1: EVs are taken up by endothelial cells. Purified EVs were labeled in green with PKH67, and incubated for 4 h with a monolayer of endothelial cells. Untreated endothelial cells are used as a negative control. After extensive washing to remove unbound EVs, the cells were stained with phalloidin to stain actin (red) and Hoechst 33342 to stain the nucleus (blue). The cells were observed by confocal microscopy. Images were taken using a laser confocal microscope and a 63x 1.3 NA oil objective. Hoechst 33342 is excited at 405 nm and with emissions collected at 450-470 nm (blue); PKH67 is excited at 488 nm, emissions collected at 505-530 nm (green); and Phalloidin-594 is excited at 543 nm, emissions collected at 620 nm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57067/57067fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57067/57067fig2.jpg" /> 
Figure 2: Effect of EV uptake on endothelial barrier. (A) Schematic of the diffusion of rhodamine-labeled dextran: endothelial cells are grown to confluence on the top of a filter membrane, the fluorescent dextran is applied to the top chamber and diffuses over time through the membrane. The effect of EVs on permeability is assessed after the addition of EVs to the top chamber by measuring the rate of diffusion of the dextran to the bottom chamber. (B) An increasing amount of EVs are incubated with endothelial cells. The transfer of rhodamine-labeled dextran across a trans-well membrane over time allows for permeability measurements. Permeability across the endothelial layer is significantly increased after 2 h of incubation with 50 and 100 &#181;g/mL of EVs. Data represent mean (&#177; s.e.m.) from 3 experiments. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57067/57067fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57067/57067fig3.jpg" /> 
Figure 3: Titration of puromycin. The stock solution of puromycin (10 mg/mL) is diluted in RPMI 1640 supplemented with 10% heat-inactivated (56 &#176;C, 30 min) fetal calf serum, 2 mM glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin/streptomycin. A total of 15 &#181;L is added to 10 mL of medium in tube A. Then 7.5 mL of the solution from tube A is mixed to 2.5 mL of RPMI to tube B. Finally, 100 &#181;L of the dilution are added to 100 &#181;L of cells to give a final concentration of 7.5 &#181;L/mL, 5.62 &#181;L/mL and so on. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57067/57067fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57067/57067fig4.jpg" /> 
Figure 4: Generation of endothelial cells overexpressing miR451a. (A) Determination of the killing curve of puromycin on endothelial cells. Endothelial cells were incubated with puromycin at increasing doses. The viability was measured by MTS assay after 72 h. The data represent the mean (&#177; s.e.m.) of one representative experiment. (B) RNAs derived from endothelial cells overexpressing miR451a or a negative control were harvested and the miR451a transcript was quantified by Real-Time PCR. qPCR results are normalized by the 2-Ct method using U6 as a housekeeper gene and expressed as mean (&#177; s.e.m.) (n = 3 experiments). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57067/57067fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Evaluation of Extracellular Vesicle Function During Malaria Infection at JoVE.com

Evaluation of Extracellular Vesicle Function During Malaria Infection

In this work, we describe protocols to investigate the role of extracellular vesicles (EVs) released by Plasmodium falciparum infected erythrocytes. In particular, we focus on the interactions of EVs with endothelial cells.

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible ...

evaluation-of-extracellular-vesicle-function-during-malaria-infection

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

7.5K Views.  University of Fribourg. In this work, we describe protocols to investigate the role of extracellular vesicles (EVs) released by Plasmodium falciparum infected erythrocytes. In particular, we focus on the interactions of EVs with endothelial cells.

Video: Evaluation of Extracellular Vesicle Function During Malaria Infection

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria1, fungi2 and parasites3. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent4. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase5. 
Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility6.
We describe methods to isolate Neutrophil Granulocytes from peripheral human blood7 and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.

Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them ...

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

2D and 3D Chromosome Painting in Malaria Mosquitoes

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.

Chromosome painting is a useful method for studying organization of the cell nucleus and evolution of the karyotype. Here, we demonstrate an approach to isolate and amplify specific regions of interest from single polytene chromosomes that are subsequently used for two- and three-dimensional fluorescent in situ hybridization (FISH).

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Evaluation of Blood Lactate and Plasma Insulin During High-intensity Exercise by Antecubital Vein Catheterization

The measurement of metabolic and endocrinal markers during physical activity is of relevance to understanding the physiological implications of different exercise modalities. During some exercise modalities (e.g., high-intensity interval exercise), blood metabolites and hormonal levels change in short periods of time. In the present study, we describe a method to catheterize the antecubital vein, which allows the collection of several blood samples during exercise. Insulin and venous lactate concentrations were measured during high-intensity exercise by the application of the described method. The exercise consisted of three 30 s bouts of high-intensity exercise separated by 4 min of recovery. After the last recovery period, a Wingate test was performed. Blood samples from the antecubital vein were obtained before and after each 30 s bout and before and after the Wingate test. As a result, it was possible to evaluate the plasma insulin and venous blood lactate variations during the exercise.

Here we present a protocol to obtain several blood samples from the antecubital vein during high-intensity interval training. This protocol may be useful for the measurement of blood metabolites and endocrinal markers during exercise.

The measurement of metabolic and endocrinal markers during physical activity is of relevance to understanding the physiological implications of different ...

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca2+ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca2+ signals induced by pathogens raises major technical challenges that need to be addressed.
Here, we report protocols for the detection of global and local Ca2+ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca2+ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca2+ signals during extended infection kinetics using the chemical probe Fluo-4.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized.

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ ...

Mouse Body Temperature Measurement Using Infrared Thermometer During Passive Systemic Anaphylaxis and Food Allergy Evaluation

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature readings is common, and they have been proven to be accurate and invaluable in this regard. However, this method of temperature measurement requires the mice to be anesthetized in order to insert the probe without injury to the animal. This limits the ability to observe other phenotypes of the mouse simultaneously. In order to investigate other phenotypes while measuring temperatures, rectal probes are not ideal, and another method is desired. Here, we introduce a noninvasive method of temperature measurement that foregoes the requirement for mouse anesthesia while maintaining equal reliability to rectal probes in measuring body temperature. We use an infrared thermometer that detects body surface temperatures at ranges between 2 and 150 mm. This method of body temperature measurement is successful in reliably replicating temperature change trends during passive system anaphylaxis experiments in mice. We show that body surface temperatures are about 2.0 &#176;C lower than rectal probe measurements, but the degree of temperature drop follows the same trend. Furthermore, we use the same technique to observe mice in a food allergy model to evaluate temperature and activity levels simultaneously.

Here we present a new method to accurately measure body temperature differences in passive systemic anaphylaxis (PSA) and food allergy mouse models using an infrared thermometer. This procedure has been accurately duplicated in previous PSA results.

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature ...

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. Immunofluorescence microscopy and image-based quantification methods remain important tools to quantitate neutrophil extracellular trap formation. However, there are key limitations to the immunofluorescence-based methods that are currently available for quantifying NETs. Manual methods of image-based NET quantification are often subjective, prone to error and tedious for users, especially non-experienced users. Also, presently available software options for quantification are either semi-automatic or require training prior to operation. Here, we demonstrate the implementation of an automated immunofluorescence-based image quantification method to evaluate NET formation called NETQUANT. The software is easy to use and has a user-friendly graphical user interface (GUI). It considers biologically relevant parameters such as an increase in the surface area and DNA:NET marker protein ratio, and nuclear deformation to define NET formation. Furthermore, this tool is built as a freely available app, and allows for single-cell resolution quantification and analysis.

Here, we present a protocol for generating neutrophil extracellular traps (NETs) and operating NETQUANT, a fully automatic software option for quantification of NETs in immunofluorescence images.

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

Evaluation of Extracellular Vesicle Function During Malaria Infection

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Neutrophil Extracellular Traps: How to Generate and Visualize Them

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

2D and 3D Chromosome Painting in Malaria Mosquitoes

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Evaluation of Blood Lactate and Plasma Insulin During High-intensity Exercise by Antecubital Vein Catheterization

Imaging Ca²⁺ Responses During Shigella Infection of Epithelial Cells

Mouse Body Temperature Measurement Using Infrared Thermometer During Passive Systemic Anaphylaxis and Food Allergy Evaluation

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Purification of High Yield Extracellular Vesicle Preparations Away from Virus