eoe sub articles

EoE Sub Articles

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Parasite culture
NOTE: Follow the local regulations for human pathogens handling.
<ol>
	<li>Maintain&#160;Plasmodium falciparum&#160;parasites according to the standard protocol as described before&#160;in the parasite culture medium (see&#160;Table of Materials).</li>
	<li>Keep the parasites tightly synchronized using sorbitol treatment as previously described.</li>
	<li>To ensure VAR2CSA expression on the surface of the Plasmodium falciparum-infected erythrocytes (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 IgG) coupled to protein G-magnetic beads.
	<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 described.</li>
	</ol>
	</li>
	<li>To verify VAR2CSA expression on the selected parasites perform flow cytometry as previously described.
	<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 described. 
	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 before. Late-stage purification can also be performed using a 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&#160;x g&#160;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 Claricep Flash Silica, CS-column coupled to a magnet (see&#160;Table of Materials) and let it slowly pass through the column. Trophozoite-IEs are paramagnetic (due to the presence of hemozoin) and will stay in 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&#160;x g&#160;for 10 min at room temperature. Carefully discard the supernatant and re-suspend it 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&#160;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&#160;IEs/mL is 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 leukemia&#160;and can be purchased from the American Type Culture Collection (ATCC). Maintain the cell line according to the provider&#39;s instructions in the THP-1 culture medium (see&#160;Table of Materials).
<ol>
	<li>For regular maintenance, start THP-1 cell culture at 2-4 x 105&#160;cells/mL and subculture when cell concentration reaches 8 x 105&#160;cells/mL. 
	NOTE: Do not allow the cell concentration to exceed 1 x 106&#160;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&#160;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 described.
	<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% fetal bovine serum (FBS) in phosphate-buffered saline (PBS).</li>
		<li>Wash three times with 2% FBS in PBS and measure surface staining by flow cytometry.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
		NOTE: The THP-1 cells are negative for CD16 and positive for CD32 and CD64 as previously reported&#160;(Figure 1).</li>
	</ol>
	</li>
	<li>For the phagocytosis assay, set up a THP-1 cell culture flask with 2.5 x 105&#160;cells/mL the day before the experiment to yield around 5 x 105&#160;cells/mL on the day of assay.&#160;&#160;&#160;&#160;&#160; 
	NOTE: Ensure 4-6 x 105&#160;cells/mL are present on the day of the assay.</li>
</ol>
3. Phagocytosis assay (Figure 2)
<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.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: Label plate 1 as an opsonization plate and use it both for ethidium bromide (EtBr) staining and opsonization. Label plate 2 as a 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&#160;IEs/mL by adding parasite culture medium and ethidium bromide&#160;(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 of 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 3A). 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&#160;x g&#160;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&#160;x g&#160;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 the 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 a parasite culture medium.</li>
		<li>Always include the following controls (Figure 3B): 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&#160;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 antibodies (data not shown). However, it is recommended to rule out variations between batches by testing several dilutions every time a new batch of antibodies 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&#160;x g&#160;for 5 min at room temperature), decant the supernatant, and re-suspend the pellet in the THP-1 cell culture medium.</li>
		<li>Spin again (500&#160;x g&#160;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&#160;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 it back in the cell culture incubator (Figure 3C).</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&#160;x g&#160;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&#160;x g&#160;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 3D)&#160;&#160;&#160; 
		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% carbon dioxide (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&#160;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&#160;x g&#160;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&#160;x g&#160;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&#160;is not&#160;recommended, since the percentage of EtBr+&#160;THP-1 cells decays rapidly (Figure 4).</li>
	</ol>
	</li>
</ol>
4. Flow cytometry acquisition and analysis&#160;&#160;&#160;&#160;&#160;
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 where 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 an FSC vs. SSC density plot, using the wells where no IEs were added (Figure 5A). 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 5B).</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+&#160;THP-1 cells) or as relative phagocytosis calculated as the percentage using the positive control as maximum.</li>
</ol>

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

an assay for quantifying antibody-mediated phagocytosis of parasite-infected erythrocytes

Source: Quintana, M. d. P. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61538">Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay.</a>&#160;J. Vis. Exp.&#160;(2020)
This video demonstrates a flow cytometry-based phagocytosis assay involving labeled and opsonized Plasmodium falciparum-infected erythrocytes by monocytes. Flow cytometry identifies fluorescence in monocytes, confirming the successful identification of opsonized erythrocytes through phagocytosis.

<img alt="Figure 1" src="/files/ftp_upload/21901/21901_Figure1.jpg" /> 
Figure 1: Fc receptors expressed on the THP-1 cell surface. (A) Fc&#947;-receptor III/CD16 (red). (B) Fc&#947;-receptor II/CD32 (green).&#160;C.&#160;Fc&#947;-receptor I/CD64 (orange). Unlabeled cells are shown in blue.
<img alt="Figure 2" src="/files/ftp_upload/21901/21901_Figure2.jpg" /> 
Figure 2: Phagocytosis assay flow chart.&#160;Flow chart depicting the main steps of the assay.
<img alt="Figure 3" src="/files/ftp_upload/21901/21901_Figure3.jpg" /> 
Figure 3: 96 well plate experiment layout.&#160;(A) Layout for IEs EtBr labeling. (B) Layout for opsonization; 6 wells are always reserved for controls. (C) Layout for THP-cells plating. (D)Layout for phagocytosis.
<img alt="Figure 4" src="/files/ftp_upload/21901/21901_Figure4.jpg" /> 
Figure 4: Color coding as in Figure 3.&#160;(A) Flow cytometry histogram overlay of one experiment acquired immediately and (B) after storage at 4 &#176;C for 12 h. (C) Percentage of EtBr+&#160;THP-1 cells measured before and after storage. NF## represents different malaria-exposed female donors.&#160;
<img alt="Figure 5" src="/files/ftp_upload/21901/21901_Figure5.jpg" /> 
Figure 5: Flow cytometry gating strategy. (A) THP-1 cells gated on FSC/SSC.&#160;(B) Ethidium bromide positive (EtBr+) THP-1 cells in an FL3 histogram. THP-1 cells alone/no IEs added (blue), THP-1 cells incubated with un-opsonized IEs (green), and THP-1 cells incubated with IEs opsonized with a positive control (red) are shown.

An Assay for Quantifying Antibody-Mediated Phagocytosis of Parasite-Infected Erythrocytes

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Parasite culture
NOTE: Follow the ...

Take Plasmodium falciparum-infected erythrocytes. Label the parasite DNA with a fluorescent dye for parasite visualization.
Incubate these parasite-infected erythrocytes with sera from malaria-exposed individuals. These cells display parasite antigens on their surface, which attach to naturally acquired human IgG antibodies in sera, opsonizing cells for phagocytosis.
Transfer the opsonized parasite-infected erythrocytes to a protein-coated multi-well plate containing a suspension of human-derived monocytes &#8212; or phagocytic cells.
During incubation, the Fc-gamma receptors on monocytes interact with the IgG-opsonized erythrocytes, triggering the engulfment of erythrocytes into phagosomes.
Post-incubation, centrifuge the plate to prevent further cellular interactions and stop phagocytosis.
Discard the supernatant. Treat with ammonium chloride lysing solution, selectively rupturing non-phagocytosed infected erythrocytes without affecting monocytes.
Add serum-containing buffer to stop the lysing process. Centrifuge and discard the supernatant containing lysed erythrocytes.
Resuspend the monocytes in a buffer.
Perform flow cytometry to quantify monocytes that display fluorescence from the parasite-infected erythrocytes within them, confirming their antibody-mediated phagocytosis.

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Nanyang Technological University

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110 Views. Source: Quintana, M. d. P. et al., Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay. J. Vis. Exp. (2020) This video demonstrates a flow cytometry-based phagocytosis assay involving labeled and opsonized Plasmodium falciparum-infected erythrocytes by monocytes. Flow cytometry identifies fluorescence in monocytes, confirming the successful identification of opsonized erythrocytes through phagocytosis. 

Video: An Assay for Quantifying Antibody-Mediated Phagocytosis of Parasite-Infected Erythrocytes - Concept

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

JoVE Journal

Immunology and Infection

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

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 ...

An Assay for Quantifying Antibody-Mediated Phagocytosis of Parasite-Infected Erythrocytes

Transcript

An Assay for Quantifying Antibody-Mediated Phagocytosis of Parasite-Infected Erythrocytes

Use of a Monocyte Monolayer Assay to Evaluate Fcγ Receptor-mediated Phagocytosis

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

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