The authors thank the Canadian Blood Services for a Graduate Fellowship Program Award to T.N.T. This research received financial support from the Canadian Blood Services' Centre for Innovation, funded by the federal government (Health Canada) and the provincial and territorial ministries of health. The views herein do not reflect the views of the federal, provincial, or territorial governments in Canada.

Obtain approval for the use of human samples by the research ethics board/institutional review board, and obtain signed consent from all human donors. The MMA can also be performed using mouse PBMCs in a similar manner as the human assay, following animal use ethics approval. The MMA utilizes aseptic tissue culture techniques.
NOTE: See Figure 1.
NOTE: Although we recommend use of a level 2 biocontainment cabinet during the assay to maintain aseptic technique, as the method is only a &#8220;short-term&#8221; culture method, there isn&#8217;t really enough time for bacteria to contaminate the assay. Therefore, if a level 2 biocontainment cabinet doesn&#8217;t exist, rather than buy one just for this assay, the assay could be performed outside of a biocontainment cabinet, on an open bench. The use of a 37 &#176;C incubator with 5% CO2, however, is not an option and must be used for optimal results.
1. Peripheral Blood Mononuclear Cell Isolation
<ol>
	<li>Obtain human whole blood from a healthy donor or a patient via venipuncture using vacutainers containing acid-citrate-dextrose (ACD) anticoagulant (yellow-top tubes). 
	NOTE: Whole blood can be stored in ACD at room temperature (18-22 &#176;C) for up to 36 h before proceeding to the next step11. Normally, 1-2 10-mL vacutainer tubes of whole blood are sufficient for the assay.</li>
	<li>Dilute the whole blood 1:1 v/v in warm complete RPMI medium (RPMI-1640 supplemented with 10% fetal bovine serum, 20 mM HEPES, and 0.01 mg/mL gentamicin).</li>
	<li>Isolate peripheral blood mononuclear cells (PBMCs) from the diluted whole blood using density gradient centrifugation, as recommended by the manufacturer (See List of Materials). Layer the diluted blood very slowly over the density gradient (warmed to room temperature, 18-22 &#176;C). 
	NOTE: Minimize the amount of mixing at the interface for the optimal separation of blood by carefully layering the blood mixture in a dropwise fashion or by using a pipette.
	<ol>
		<li>Allow the blood mixture to slowly layer over the top of the density gradient by placing the pipet tip close to the density gradient and by enabling the blood mixture to run down the side of the tube very slowly.</li>
		<li>Depending on the scale of experiment, layer 10 mL of blood mixture on top of 3 mL of density gradient (in a 15-mL tube) or layer 35 mL of blood mixture on top of 15 mL of density gradient (in a 50-mL tube). Typically, 10 mL of whole blood yields 10 million PBMCs, with some donor-to-donor variation. 
		NOTE: It is very important that there is no mixing of the blood mixture with the density gradient. The blood mixture should layer over the density gradient and slowly rise until all the blood is on top of the density gradient.</li>
		<li>Centrifuge the layered mixture at 700 x g for 30 min without brakes. The centrifuged mixture should separate into 5 layers (from top to bottom): plasma, buffy coat (containing PBMCs), density gradient material, granulocytes, and RBCs.</li>
		<li>Remove and discard the majority of the plasma and carefully retrieve the buffy coat (PBMC) content into a new 15-mL tube using a Pasteur pipette and a suction bulb. 
		NOTE: The removal of the buffy coat layer is effectively done by applying suction on the Pasteur pipette while performing a circular motion around the outside of the layer, with the tip of the pipette against the tube.</li>
	</ol>
	</li>
	<li>Wash the isolated PBMCs three times in pH 7.3 phosphate-buffered saline (PBS) by centrifuging at 350 x g for 10 min (with full brakes) in between washes. Reconstitute the PBMC pellet in complete RPMI medium. Depending on the size of the pellet, 3-7 mL of medium is sufficient.</li>
	<li>Count the PBMC using trypan blue and a hemocytometer. Only count those cells that are not stained by the trypan blue. Reconstitute the PBMCs to 1,750,000 cells/mL in complete RPMI medium.</li>
	<li>Seed 400 &#181;L (700,000 cells) into each well of an 8-chamber slide. Incubate the slide in a 37 &#176;C, fully humidified tissue culture incubator (supplemented with 5% CO2) for 1 h to allow the monocyte/macrophages to adhere.</li>
</ol>
2. Pre-treatment of Adhered Monocytes
NOTE: This step is only necessary if looking for ways to inhibit or enhance phagocytosis.
<ol>
	<li>Pre-treat adhered monocytes with any drug(s) or compound(s) of interest. Reconstitute the drug(s) or other test material to the desired concentration using complete RPMI medium. 
	NOTE: For example, 200 &#181;g/mL of IVIG is typically used to yield a 95-100% inhibition of phagocytosis when using human monocytes.</li>
	<li>Aspirate and discard the supernatant containing any non-adherent cells from the 8-chamber slide after the 1-h incubation (after step 1.6). Replace it with 400 &#181;L of a drug or another treatment and incubate for 1 h at 37 &#176;C.
	<ol>
		<li>When aspirating and replacing solutions in the 8-chamber slide, make sure to control the flow of the fluids so that weakly adhered cells do not lift off. Also, work with only 2-3 empty wells at a time and avoid drying the wells. 
		NOTE: Typically, each treatment is performed in technical triplicates.</li>
	</ol>
	</li>
</ol>
3. Opsonization of R2R2 Red Blood Cells
NOTE: The R2R2 used here are obtained from the Blood Collection Center (Canadian Blood Services), but they are also commercially available. Opsonized R2R2 RBCs are used as a positive control for Fc&#947;R-mediated phagocytosis. Na&#239;ve R2R2 should be stored in Alsever&#39;s solution (to prolong shelf life) at 4 &#176;C for up to 1 month. Alsever&#39;s solution is made in-house and composed of 0.8% w/v trisodium citrate (dihydrate), 1.9% w/v dextrose, 0.42% w/v sodium chloride, and 0.05% w/v citric acid (monohydrate). If R2R2 cells are not available, other Rh-phenotyped cells, such as R1R2, R1R1, R1r, or R2r, can be used.
<ol>
	<li>Wash R2R2 (cDE/cDE) red blood cells in PBS a total of three times using centrifugation at 350 x g for 5 min each. 
	NOTE: The amount of RBCs needed depends on the experimental size and can be back-calculated. Always wash an excess amount of RBCs, since RBCs are lost during each wash due to lysis or during the removal of supernatant. For example, in an experiment with 5 treatments and 2 controls, all conducted in technical triplicates, there is a total of 21 wells. 21 wells with 400 &#181;L/well of 1.25% RBC mixture indicates that 105 &#181;L of packed, opsonized RBC are needed. 200 &#181;L of RBC should be initially washed and 150 &#181;L should be opsonized to ensure that there is an adequate amount of RBCs for the phagocytosis step.</li>
	<li>Opsonize the washed R2R2 pellet with 1:1 v/v polyclonal anti-D antibodies from human serum and incubate for 1 h at 37 &#176;C, with intermittent mixing. 
	NOTE: If polyclonal anti-D antibodies are not available, it is possible to use monoclonal anti-D antibodies, which are commercially available, or anti-D that is normally used for Rh immune prophylaxis, which can be obtained from blood banks or transfusion services. Optimization testing should be conducted at the beginning, when setting up the assay, and whenever switching lots of un-titered polyclonal antibodies or switching to a monoclonal antibody. This is to identify the optimal concentration or volume of anti-D required for an antiglobulin test (IAT) result of between 3+ and 4+ and a phagocytosis result of between 70-90 phagocytosed RBCs per 100 monocytes counted.</li>
	<li>Wash the opsonized R2R2 a total of three times in PBS using centrifugation at 350 x g for 5 min each. 
	NOTE: Successful opsonization of R2R2 can be confirmed by performing an indirect IAT. Briefly, secondary polyclonal anti-human antibodies are added to bind primary opsonizing antibodies on RBC surfaces, and the amplified signal can be observed in the form of hemagglutination. A detailed manufacturer protocol can be found in the supplementary materials file.</li>
	<li>Reconstitute the washed R2R2 pellet to 1.25% v/v using complete RPMI medium. 
	NOTE: Excess opsonized R2R2 can be stored in Alsever&#39;s solution at 4 &#176;C for up to a week.</li>
</ol>
4. Fc Receptor-mediated Phagocytosis
<ol>
	<li>Aspirate the drug or medium supernatant from the 8-chamber slide and add 400 &#181;L of the 1.25% v/v R2R2 mixture. Incubate at 37 &#176;C for 2 h.</li>
	<li>After 2 h of incubation, remove the chambers using the manufacturer&#39;s adaptors. Dab off excess R2R2 on a paper towel. Make sure that the slide does not dry out.</li>
	<li>Fill a 100-mL beaker with PBS. Submerge and wash the slide by slowly moving the slide back and forth (around 30-40 strokes) to remove the majority of the un-phagocytosed R2R2.</li>
	<li>Remove the slide from the PBS. Dab off excess PBS using a paper towel or tissue and air-dry the slide.</li>
	<li>Fix the air-dried slide in 100% methanol for 45 s. Then, air-dry the fixed slide. 
	NOTE: Slides can be fixed using another method that is more compatible for downstream staining, such as the Gr&#252;nwald-Giemsa stain21 or the Wright-Giemsa stain22.</li>
	<li>Mount the slide using an in-house-made elvanol mounting medium (or another commercially available mounting medium) and add coverslips. 
	NOTE: Elvanol mounting medium is composed of 15% w/v polyvinyl alcohol resin and 30% v/v glycerin in PBS. The mixture is heated until all the resin has dissolved and the glycerin is homogenously mixed. This can be replaced with other commercially available mounting medium.</li>
	<li>Allow the mount to dry overnight before quantification.</li>
</ol>
5. Quantification of Phagocytosis
<ol>
	<li>Using a phase-contrast microscope and a 40X objective lens, manually quantify the amount of phagocytic events by counting at least 200 monocytes and the number of phagocytosed R2R2 within these monocytes. Have one counter in each hand to simultaneously quantify the number of monocytes and the number of phagocytosed R2R2.</li>
	<li>Obtain the average phagocytic index by dividing the number of phagocytosed R2R2 by the number of monocytes and multiplying by 100. Express the data as the mean (average phagocytic index) &#177; the standard error of the mean (SEM).</li>
</ol>

<ol>
	<li>Hunt, J. S., Beck, M. L., Hardman, J. T., Tegtmeier, G. E., Bayer, W. L. <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+human+erythrocyte+alloantibodies+by+IgG+subclass+and+monocyte+interaction.">Characterization of human erythrocyte alloantibodies by IgG subclass and monocyte interaction.</a> Am J Clin Pathol. 74 (3), 259-264 (1980).</li><li>Schanfield, M. S., Stevens, J. O., Bauman, 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+detection+of+clinically+significant+erythrocyte+alloantibodies+using+a+human+mononuclear+phagocyte+assay.">The detection of clinically significant erythrocyte alloantibodies using a human mononuclear phagocyte assay.</a> Transfusion. 21 (5), 571-576 (1981).</li><li>Branch, D. R., Gallagher, M. T., Mison, A. P., Sysiokian, A. L., Petz, L. D. <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+determination+of+red+cell+alloantibody+significance+using+an+assay+of+monocyte-macrophage+interaction+with+sensitized+erythrocytes.">In vitro determination of red cell alloantibody significance using an assay of monocyte-macrophage interaction with sensitized erythrocytes.</a> Br J Haematol. 56 (1), 19-29 (1984).</li><li>Hunt, J. S., Beck, M. L., Wood, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte-mediated+erythrocyte+destruction.+A+comparative+study+of+current+methods.">Monocyte-mediated erythrocyte destruction. A comparative study of current methods.</a> Transfusion. 21 (6), 735-738 (1981).</li><li>Noumsi, G. T., Billingsley, K. L., Moulds, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+transfusion+of+antigen+positive+blood+to+alloimmunised+patients+using+a+monocyte+monolayer+assay.">Successful transfusion of antigen positive blood to alloimmunised patients using a monocyte monolayer assay.</a> Transfus Med. 25 (2), 92-100 (2015).</li><li>Moulds, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+antibodies+and+complement.">Introduction to antibodies and complement.</a> Transf Apher Sci. 40 (3), 185-188 (2009).</li><li>Grandstaff Moulds, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+identification.">Antibody identification.</a> Transf Apher Sci. 40 (3), 195-197 (2009).</li><li>Hendrickson, J. 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J., Sterling, 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+tagging+of+red+cells+and+plasma+proteins+with+radioactive+chromium.">The tagging of red cells and plasma proteins with radioactive chromium.</a> J Clin Invest. 29 (12), 1604-1613 (1950).</li><li>Mollison, P. L., Veall, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+the+isotope+51Cr+as+a+label+for+red+cells.">The use of the isotope 51Cr as a label for red cells.</a> Br J Haematol. 1 (1), 62-74 (1955).</li><li>Sebring, E. S., Polesky, H. 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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=Small+molecule+phagocytosis+inhibitors+for+immune+cytopenias.">Small molecule phagocytosis inhibitors for immune cytopenias.</a> Autoimmun Rev. 15 (8), 843-847 (2016).</li><li>Fitzer-Attas, C. 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=Fcgamma+receptor-mediated+phagocytosis+in+macrophages+lacking+the+Src+family+tyrosine+kinases+Hck,+Fgr,+and+Lyn.">Fcgamma receptor-mediated phagocytosis in macrophages lacking the Src family tyrosine kinases Hck, Fgr, and Lyn.</a> J. Exp Med. 191 (4), 669-682 (2000).</li><li>Allhorn, 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=The+IgG-specific+endoglycosidase+EndoS+inhibits+both+cellular+and+complement-mediated+autoimmune+hemolysis.">The IgG-specific endoglycosidase EndoS inhibits both cellular and complement-mediated autoimmune hemolysis.</a> Blood. 115 (24), 5080-5088 (2010).</li><li>Li, 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=Inhibition+of+phagocytic+recognition+of+anti-D+opsonized+Rh+D++RBC+by+polymer-mediate+immunocamouflage.">Inhibition of phagocytic recognition of anti-D opsonized Rh D+ RBC by polymer-mediate immunocamouflage.</a> Am J Hematol. 90 (12), 1165-1170 (2015).</li><li>Kwan, D. 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The authors have nothing to disclose.

The MMA is a laborious technique that requires expertise in both tissue culture and microscopy. There are several critical steps to ensure success: 1) generation of the monocyte monolayer; 2) opsonization of the RBCs, and 3) manual quantification. The monocyte monolayer does not adhere very strongly to the chamber slide, so physiological pH must be maintained throughout the assay11 and an adequate number of PBMCs should be seeded. Vigorous pipetting, which might disturb the adhered cells, should be avoided. One approach is to always remove and add solutions from the same corner of the chamber and to ensure that the motion is slow and steady. Similarly, during the last washing step to remove the excess RBCs, the movement should be slow and steady. This ensures minimal disturbance to the monolayer while still removing the majority of the un-phagocytosed RBCs. Inadequate washing will lead to a high background of contaminating RBCs, which makes manual quantification difficult. Secondly, the R2R2 RBCs must be sufficiently opsonized to obtain an average phagocytic index of 80-120 for the phagocytosis control. This desired phagocytic range strikes a balance between the ease of counting (e.g., monocytes with more than 5 phagocytosed RBCs are difficult to accurately quantify) and maintaining an adequate amount of phagocytosis for statistical analysis. The degree of opsonization can be confirmed by an IAT, and a reading between 3+ to 4+ is needed. The R2R2 RBCs should be discarded when there is excess lysis during washing, when the supernatant turns dark red, or when a significant decrease in phagocytosis is observed in experiments due to the aging of the cells in storage. Lastly, manual quantification using the microscope can be tricky, especially when comparing counts between lab personnel and between experiments. By examining the same field on each well, or simply by counting more cells, a more consistent count can be obtained. Side-by-side training with an experienced technician and the use of a designated set of training slides is recommended.
A major critique of the MMA is the subjectivity of the manual quantification step. However, with adequate training, consistency can be obtained across different counters. Another limitation is the intrinsic donor-to-donor differences in monocyte phagocytic abilities and in R2R2 surface antigen expression levels, which is a source of data variation when dealing with human samples.
Other alternative techniques are available for examining Fc&#947;R-mediated phagocytosis. The majority of commercial kits utilize fluorescent output to monitor phagocytosis (e.g., bioparticles, pH-sensitive fluorescence protein, or IgG-labeled fluorescent latex beads). The use of fluorescent output does offer more objective quantification, but one also needs to consider the availability, cost, and training associated with the use of a fluorescent microscope or a flow cytometer, as well as the ensuing dependence upon commercially available kits.
Finally, this assay can be modified depending upon the research question. For example, when testing drug inhibition of phagocytosis, monocytes can either be pre-treated or co-incubated with both the drugs and opsonized RBCs (i.e., a competition assay). The downstream signaling of antibodies of different subtypes, chimeric antibodies, or recombinant constructs can also be tested. With recent breakthroughs in the development of a universal antigen-null blood24, the MMA could be utilized in initial screens of these antigen-null RBCs with various antibodies to assess whether there is indeed a lowered efficacy in triggering phagocytosis.

The monocyte monolayer assay (MMA) is an in vitro assay originally developed to better predict blood transfusion outcomes in patients with auto- or alloantibodies to red blood cells (RBCs)1-5. By assessing the effect of anti-RBC antibodies in mediating Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis using this in vitro assay, it is possible to predict the clinical outcome in vivo. Indeed, the MMA has been used successfully to avoid immune destruction of antibody-bound RBCs, despite the transfusion of serologically incompatible blood5. The typical pre-transfusion procedure for compatibility testing, also termed crossmatching, involves serological methods that include typing the patient's blood for ABO and Rh antigens and screening for the presence of anti-RBC antibodies in the patient6. Blood matched for ABO/Rh is selected, and if antibodies are present, an attempt to identify them is made so that blood for transfusion can be further selected to avoid these antigens. An ideal crossmatch result occurs when all donor blood is serologically compatible with the patient's blood, which reduces the risk of post-transfusion hemolysis7. However, this system falls short for the small group of patients who have become alloimmunized upon repeated transfusion or pregnancy. These patients produce alloantibodies against specific RBC antigens. Some produce antibodies to antigens of very high frequency in the general population, and thus become progressively more difficult to crossmatch8,9. Adding to the complexity, not all alloantibodies are clinically significant; in other words, the binding of an alloantibody to RBCs detected by a serology test does not necessarily result in hemolysis when antigen-positive, incompatible blood is transfused. The MMA was originally developed to assess the potential clinical significance of serologically incompatible blood in a transfusion setting1-5.
Since extravascular hemolysis of antibody-bound RBCs is known to be mediated by the mononuclear phagocyte system, primary monocytes/macrophages are utilized in the development of diagnostic assays. The first assay to study the interaction of monocytes, RBCs, and antibodies was published in 1975, but the sub-optimal conditions used led only to rosette formation (the binding of RBCs to the periphery of the monocyte), and no phagocytosis was observed10. Significant modifications to the assay were made by several groups, leading to an assay for which the level of phagocytosis of alloantibody-bound RBCs could be correlated to the clinical outcome of hemolysis1-5. Recently, the optimal storage conditions of clinical samples and further optimization of assay conditions were examined to enhance the utility of a clinical MMA crossmatch using autologous patient samples11.
Three other diagnostic techniques have been employed in addition to the MMA in predicting transfusion outcomes: the 51Cr release test, the rosette test, and the chemiluminescence test (CLT). In the 51Cr release test, the patient is injected with 51Cr-labeled donor RBCs, and the half-life of the labeled RBCs is monitored and is predictive of post-transfusion survival or clearance12,13. As this method uses radioactive materials, it is rarely performed anymore. The rosette test involves mixing and incubating monocytes with RBCs and quantifying the level of rosette formation (with no phagocytosis)14. The clinical significance of antibodies in vivo involves active phagocytosis by macrophages found in the spleen and/or the liver; thus, this method does not provide a relevant readout of phagocytosis. The CLT uses luminol to monitor the oxidative burst during monocyte phagocytosis of RBC, since luminol fluoresces blue when oxidized in the phagosome15. This method is good, but contamination by neutrophils can confound the readout. Parallel comparisons have been made to evaluate the sensitivity, practicality, and reproducibility of the four available methods, and both the CLT and MMA were ranked superior16. However, the CLT has been mainly utilized in assessing hemolytic disease of the fetus and newborn (HDFN), and the assay's optimal pH of 8.0 might compromise the level of phagocytosis11.
In addition to its diagnostic and clinical utility, the MMA has been modified for other research purposes. Indeed, the MMA can not only serve as a functional assay to address discrepancies between serology and biology, it has also been used to retrospectively investigate the cause of hemolysis after intravenous immunoglobulin (IVIG) therapy17. It has also been used to examine the structure-function of chemical inhibitors of Fc&#947;R-mediated phagocytosis18-20 and to study the downstream signaling of Fc&#947;R-mediated phagocytosis21. In our laboratory, in addition to using a human MMA, we are developing a murine MMA using primary mouse peripheral blood mononuclear cells (PBMCs) and autologous RBCs. The rationale is to screen antibodies that can induce Fc&#947;R-mediated phagocytosis as an intermediate to developing an in vivo autoimmune hemolytic anemia (AIHA) mouse model (unpublished data). The various modifications focus on different aspects of the IgG antibody and Fc&#947;R interaction that induce phagocytosis.

<table><tbody><tr><td>Acid citrate dextrose (ACD) vacutainers</td><td>BD</td><td>REF364606</td><td></td></tr><tr><td>RPMI 1640</td><td>Sigma</td><td>R8758</td><td></td></tr><tr><td>HEPES</td><td>Bioshop</td><td>HEP003.100</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Multicell&amp;nbsp;</td><td>080150</td><td></td></tr><tr><td>Gentamicin</td><td>Gibco</td><td>15710-64</td><td></td></tr><tr><td>Ficoll-Paque PLUS</td><td>GE</td><td>17-1440-03</td><td>https://www.gelifesciences.com/</td></tr><tr><td>Phosphate buffered saline</td><td>Sigma</td><td>D8537</td><td></td></tr><tr><td>8-chamber slides</td><td>Lab-Tek-ll</td><td>154534</td><td></td></tr><tr><td>R&lt;sub&gt;2&lt;/sub&gt;R&lt;sub&gt;2&lt;/sub&gt; (cDE/cDE) red blood cells</td><td>Canadian Blood Services</td><td></td><td>Commercially available (&lt;em&gt;e.g.&lt;/em&gt;, http://www.bio-rad.com/en-ca/product/reagent-red-blood-cells)</td></tr><tr><td>Polyclonal anti-D from human serum</td><td>Gamma Biologics</td><td>DIN 02247724</td><td>Can be substituted with commercially available monoclonal anti-D or with Rh immune globulin</td></tr><tr><td>100% methanol</td><td>Caledon</td><td>6700-1-42</td><td></td></tr><tr><td>Polyvinyl alcohol resin</td><td>Sigma</td><td>P8136</td><td>Can be substituted with commercially available mount</td></tr><tr><td>UltraPure glycerine</td><td>Invitrogen</td><td>15514-011</td><td></td></tr><tr><td>Cover slips</td><td>VWR</td><td>48366 067</td><td></td></tr><tr><td>Novaclone anti-IgG</td><td>Immucorgamma</td><td>5461023</td><td>Optional for IAT (http://www.fda.gov/downloads/biologicsbloodvaccines/.../ucm081743.pdf)</td></tr></tbody></table>

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.

By following the crucial steps in Figure 1 and the procedure outlined above, the MMA can be reproducibly performed. IVIG was used as an example for the inhibition of phagocytosis in Figure 2. IVIG is known to bind and block the Fc receptors, thus inhibiting the downstream outcome of phagocytosis. By titrating the amount of IVIG used, a dose-dependent inhibition is observed, in which concentrations above 200 &#181;g/mL resulted in close to 100% inhibition and concentrations below 0.5 &#181;g/mL did not inhibit at all (Figure 2A). When the phagocytic indices are transformed and normalized to the R2R2 positive control as 0% inhibition, an inhibition curve with an IC50 of 3 &#181;g/mL can be determined (Figure 2B).
In addition to performing the assay consistently, quantification of the assay can sometimes be difficult. Figure 3 is a collection of phase-contrast microscopy images of various MMA slides. With microscopy experience, one can distinguish a monocyte from a contaminating RBC (Figure 3D), or a vacuole from a phagocytosed RBC (Figure 3B). Dense clusters of cells and/or debris should be avoided during quantification (Figures 3E and F). Also, one should avoid over-opsonizing the R2R2 RBC, which would lead to heightened phagocytosis that overcrowds the monocyte interior and interferes with accurate quantification (Figure 3C).
<img alt="Figure 1" src="/files/ftp_upload/55039/55039fig1.jpg" /> 
Figure 1. Schematic diagram of the MMA. A step-by-step depiction of the crucial steps in the MMA: isolating PBMC from whole blood, feeding the adhered monocytes with opsonized R2R2, and washing the chamber slides. <a href="http://cloudflare.jove.com/files/ftp_upload/55039/55039fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55039/55039fig2.jpg" /> 
Figure 2. Intravenous IgG (IVIG) inhibits phagocytosis in vitro using the MMA. IVIG is known to inhibit phagocytosis and is used as an inhibition control in the human MMA. (A) Titration of IVIG resulted in a dose-dependent inhibition of phagocytosis when compared to the untreated R2R2 control. The results show the mean &#177; the standard error of the mean (SEM) of n = 3 experiments. The statistical analysis was conducted using Student t-test: ** P&#8804;0.01 and *** P&#8804;0.001. (B) Inhibition curve of IVIG with an IC50 of 3 &#181;g/mL (dotted line). The results show data normalized to untreated R2R2 (0% inhibition); mean &#177; SEM of n = 3 experiments. <a href="http://cloudflare.jove.com/files/ftp_upload/55039/55039fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55039/55039fig3.jpg" /> 
Figure 3. Phase-contrast microscopy images of sample slides under 40X magnification. (A) The perfect slide in which the monocytes phagocytosed 1-2 R2R2 on average (black arrows with a distinctive halo glow), with very few contaminating, un-phagocytosed R2R2 in the background (white arrows). (B) As shown in this slide, monocytes sometimes have enlarged vacuoles (white arrows), which can be mistaken for phagocytosed R2R2. (C) When R2R2 have been over-opsonized, more than 4-5 phagocytosed R2R2 per monocyte (black arrows) render accurate counting difficult, since the R2R2 are crowded within the monocyte and distinct cell boundaries cannot be distinguished. (D) Inadequate washing of the slide leads to abundant R2R2 contamination (white arrows), which could be mistaken for adhered RBCs. (E) Sometimes, monocytes and contaminating RBCs may form clusters. Clusters also indicate bacterial contamination, which should be avoided. (F) Monocytes may form larger aggregates, which should be avoided during quantification. Using random selection of the fields to view, panel A is what is usually observed if the MMA has been performed properly. Scale bar is 20 &#181;m. <a href="http://cloudflare.jove.com/files/ftp_upload/55039/55039fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Use of a Monocyte Monolayer Assay to Evaluate FcÎ³ Receptor-mediated Phagocytosis at JoVE.com

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

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 ...

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17.9K Views.  University of Toronto. The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fcγ receptor (FcγR)-mediated phagocytosis.

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

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides the necessary methodology to add this assay to an exercise immunology arsenal. The first step in this assay is to prepare two aliquots ("H" and "F") of whole blood (heparin). Then, dihydroethidium is added to the H aliquot, and both aliquots are incubated in a warm water bath followed by a cold water bath. Next, Staphylococcus aureus (S. aureus) is added to the H aliquot and fluorescein isothiocyanate-labeled S. aureus is added to the F aliquot (bacteria:phagocyte = 8:1), and both aliquots are incubated in a warm water bath followed by a cold water bath. Then, trypan blue is added to each aliquot to quench extracellular fluorescence, and the cells are washed with phosphate-buffered saline. Next, the red blood cells are lysed, and the white blood cells are fixed. Finally, a flow cytometer and appropriate analysis software are used to measure granulocyte and monocyte phagocytosis and OB activity. This assay has been used for over 20 years. After heavy and prolonged exertion, athletes experience a significant but transient increase in phagocytosis and an extended decrease in OB activity. The post-exercise increase in phagocytosis is correlated with inflammation. In contrast to normal weight individuals, granulocyte and monocyte phagocytosis is chronically elevated in overweight and obese participants, and is modestly correlated with C-reactive protein. In summary, this flow cytometry-based assay measures the phagocytosis and OB activity of phagocytes and can be used as an additional measure of exercise- and obesity-induced inflammation.

The purpose of this manuscript is to present a method for measuring monocyte and granulocyte phagocytosis and oxidative burst activity in human blood samples.

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

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 ...

Neutrophil Functional Assays for Screening Different Signaling Pathways

A Set of Screening Techniques for a Quick Overview of the Neutrophil Function

Neutrophils are known as one of the first lines of defense in the innate immune response and can perform many particular cellular functions, such as chemotaxis, reverse migration, phagocytosis, degranulation of cytotoxic enzymes and metabolites, and release of DNA as neutrophil extracellular traps (NETs). Neutrophils not only have tightly regulated signaling themselves, but also participate in the regulation of other components of the immune system. As fresh neutrophils are terminally differentiated, short-lived, and highly variable among individuals, it is important to make the most of the collected samples. Researchers often need to perform screening assays to assess an overview of the many neutrophil functions that may be affected by specific conditions under evaluation. A set of tests following a single isolation process of normal density neutrophils was developed to address this need, seeking a balance between speed, comprehensiveness, cost, and accuracy. The results can be used to reason and guide in-depth follow-up studies. This procedure can be carried out in an average time of 4 h and includes the evaluation of cell viability, reactive oxygen species (ROS) production, real-time migration, and phagocytosis of yeast on glass slides, leaving enough cells for more detailed approaches like omics studies. Moreover, the procedure includes a way to easily observe a preliminary suggestion of NETs after fast panoptic staining observed by light microscopy, with a lack of specific markers, albeit enough to indicate if further efforts in that way would be worthwhile. The diversity of functions tested combines common points among tests, reducing the analysis time and expenses. The procedure was named NeutroFun Screen, and although having&#160;limitations, it balances the aforementioned factors. Furthermore, the aim of this work is not a definite test set, but rather a guideline that can easily be adjusted to each lab&#39;s resources and demands.

Author Spotlight: Balancing Speed, Cost, and Accuracy in Neutrophil Function Assessment with NeutroFun Screen Protocol 

This protocol features a set of neutrophil functional assays to be used as a screening method to cover functions from different signaling pathways. The protocol includes an initial and simple evaluation of cell viability, purity, reactive oxygen species production, real-time migration, phagocytosis, and a preliminary suggestion of neutrophil extracellular traps.

Neutrophils are known as one of the first lines of defense in the innate immune response and can perform many particular cellular functions, such as ...

Prediction of Red Blood Cell Antibody Significance Using the Monocyte-Macrophage Assay

Derived from monocytes in the bone marrow, macrophages are large, innate immune cells that play a major role in clearing dead cells, debris, tumor cells, and foreign pathogens. The phagocytic capacity of monocytes versus macrophages is a concept that is not well understood. Here, we aim to examine a difference in the phagocytosis of monocytes versus macrophages, specifically M1/M2 macrophages, against various opsonized red cells using a modified and updated version of the established monocyte monolayer assay (MMA). Peripheral blood mononuclear cells (PBMCs) were isolated from donor buffy coats. Using purified monocytes, inflammatory M1 and anti-inflammatory M2 macrophages were produced by in vitro culture and polarization. M1/M2 cells were harvested and used in an MMA-like assay, which we refer to as the M-MA, to decipher clinically significant phagocytosis of various red cell antibodies. A phagocytic index (PI) &#62; 5 was deemed clinically significant phagocytosis with the use of monocytes. A phagocytic index (PI) &#62; 12 was deemed clinically significant phagocytosis with the use of M1/M2 macrophages. M2 macrophages demonstrate an increased ability to phagocytose opsonized RBCs compared to monocytes and M1s. The same weak antibody (anti-S) yields significant phagocytosis with only M2 macrophages (PI=43) but not M1s (PI=2) or monocytes (PI=0), and this was demonstrated repeatedly using various antibodies. The use of M2 macrophages instead of monocytes may allow for more accurate results as these cells are more phagocytic, offering further clinical relevance to the assay. Further studies with different antibodies to red blood cells, including validation of the monocyte-macrophage assay (M-MA) with antibodies having known clinical significance, may show the M-MA&#160;more useful to help predict clinically significant red cell alloantibodies and transfusion reactions. This method will advance the field of transfusion medicine and immunology.

We have developed enhancements and updated methods for the existing monocyte-monolayer assay (MMA), in which macrophages are used to help better predict the clinical relevance of red cell alloantibodies in transfusion medicine and immunology. This assay is named the monocyte-macrophage assay (M-MA).

Derived from monocytes in the bone marrow, macrophages are large, innate immune cells that play a major role in clearing dead cells, debris, tumor cells, ...

An In Vitro Assay for Evaluating the Immunomodulatory Impact of Monocytes on Leukocyte Proliferation

Source: Hsu, P., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/53265">Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs).</a> J. Vis. Exp. (2015)
This video demonstrates an in vitro assay to explore the immunomodulatory influence of mesenchymal stem cells (MSCs) on effector T cell proliferation. The assay includes co-culturing MSCs with PBMCs to induce the monocyte population to acquire an immunomodulatory phenotype, leading to the secretion of interleukin-10 (IL-10). Following incubation with activated CD4+ T cells, the induced monocytes effectively suppress T cell proliferation.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Density Gradient Isolation of ...

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Immunology

Immunodiagnostics

In Vitro and In Vivo Models

An Assay to Assess Phagocytosis and Oxidative Burst Activity In Granulocytes and Monocytes

Source: Meaney, M. P. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/54264">Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood.</a> J. Vis. Exp. (2016)
This video demonstrates the assay to confirm phagocytic activity by cells in human blood via flow cytometry. The granulocytes and monocytes confirm their phagocytic activity by showing green and red fluorescence confirming the presence of engulfed bacteria and oxidative burst activity respectively.

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

Specialized Assays

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

Source: Quintana, M. d. P. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/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.

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

An In Vitro Assay to Measure Antibody-Dependent Cellular Phagocytosis by Phagocytes

Source: Powell, R. L., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/60149">Isolation of Leukocytes from Human Breast Milk for Use in an Antibody-dependent Cellular Phagocytosis Assay of HIV Targets.</a> J. Vis. Exp. (2019).
This video demonstrates an assay to measure the antibody-dependent cellular phagocytosis (ADCP) by breast milk phagocytes. Fluorescently labeled microspheres conjugated to an antigen-antibody complex, are combined with a cell suspension isolated from breast milk to induce antibody-dependent cellular phagocytosis by the phagocytic leukocytes. Upon labeling the cells with antibodies against common leukocyte antigen (CD45), flow cytometry is used to measure the fluorescence intensity of microsphere-positive viable phagocytic leukocytes, indicating the level of ADCP activity.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Target Microsphere preparation

	...

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

Summary

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Chapters in this video

Determining the Phagocytic Activity of Clinical Antibody Samples

Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

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

A Set of Screening Techniques for a Quick Overview of the Neutrophil Function

Prediction of Red Blood Cell Antibody Significance Using the Monocyte-Macrophage Assay

An In Vitro Assay for Evaluating the Immunomodulatory Impact of Monocytes on Leukocyte Proliferation

An Assay to Assess Phagocytosis and Oxidative Burst Activity In Granulocytes and Monocytes

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

An In Vitro Assay to Measure Antibody-Dependent Cellular Phagocytosis by Phagocytes