an in vitro assay to measure antibody-dependent cellular phagocytosis by phagocytes

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

For an ADCP assay, quickly invert the prepared ADCP plate to decant the supernatant after the final centrifugation, and add 5 x 104 freshly isolated breastmilk cells in 200 microliters of PBS plus BSA for a 2-hour incubation at 37 degrees Celsius.
At the end of the incubation, centrifuge the cells, and wash the pellets two times in 200 microliters of fresh PBS as demonstrated. After the last wash, stain the cells with 0.5 micrograms per milliliter of fixable viability stain, 450 per well, and 50 microliters of PBS for 30 minutes at room temperature, protected from light.
At the end of the incubation, pellet the cells for two washes in fresh PBS as demonstrated and stain the cells for the leukocyte markers of interest for 30 minutes at room temperature, protected from light. At the end of the incubation, collect the cells by centrifugation, and wash the cells two times in 200 microliters of 1% BSA plus PBS per wash.
After the last wash, fix the pellets in 200 microliters of 0.5% formaldehyde per well for 30 minutes at room temperature, protected from light. For flow cytometric analysis of the cell samples, set a flow cytometer equipped with a high throughput plate reader to withdraw 175 microliters of sample and to collect 5,000 cells per well.
Perform the initial gating to eliminate doublets and debris on a forward-scatter versus side-scatter plot. Use a side-scatter versus viability stain plot to eliminate the dead cells, and use a side-scatter versus CD45 plot to differentiate the major leukocyte classes.
For all CD45-positive cells, or for each leukocyte subset of interest, measure the antibody-dependent cellular phagocytosis activity by gating with a marker on the bead-positive cells in a histogram of the appropriate fluorophore channel for the conjugated beads. Then, calculate the antibody-dependent cellular phagocytosis scores as the median of the fluorescence intensity of the bead-positive cells by the percentage of the total CD45 plus the cells in the positive population.

an-vitro-assay-to-measure-antibody-dependent-cellular-phagocytosis

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

EoE Sub Articles

eoe sub articles

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Target Microsphere preparation
<ol>
	<li>Select a relevant target antigen. 
	NOTE: In this example, the recombinant protein V1V2-2F5K was used, which was designed to mimic the trimeric apex of native HIV envelope.</li>
	<li>Perform biotinylation using a commercial kit (Table of Materials) according to the manufacturer&#39;s protocol.
	<ol>
		<li>Calculate mmol of the biotin reagent to add to the reaction for a 5-fold molar excess using the formula: mmol biotin = mL protein x (mg protein/mL protein) x (mmol biotin/mg protein) x (5 mmol biotin/mmol protein). Then calculate &#181;L of the biotin reagent to add using the formula: &#181;L biotin = mmol biotin x (1,000,000 &#181;L/L) x (L/10 mmol).</li>
		<li>Equilibrate biotin to room temperature before opening. Dissolve protein in 0.5&#8722;2.0 mL of phosphate-buffered saline (PBS) according to the calculation made above.</li>
		<li>Prepare a 10 mM solution of biotin reagent in dimethylsulfoxide (DMSO) and add the appropriate volume of 10 mM biotin reagent to the protein solution, and incubate reaction on ice for 2 h or at room temperature for 30 min.</li>
	</ol>
	</li>
	<li>Remove excess biotin using protein concentrators (polyethersulfone [PES] membranes, 3 kDa molecular weight cut-off [MWCO], 0.5 mL; Table of Materials) according to the manufacturer&#39;s instructions.
	<ol>
		<li>Deposit sample into the upper chamber of spin column and add PBS up to 400 &#181;L. Cap, then insert this sample chamber into a collection tube. Centrifuge at 12,000 x g at room temperature for 30 min.</li>
		<li>Discard flow-through and add PBS to 400 &#181;L. Repeat centrifugation. Discard flow-through and add PBS to 100 &#181;L. Measure protein concentration by a spectrophotometer. 
		NOTE: Protein can be aliquoted and frozen at -80 &#176;C until used.</li>
	</ol>
	</li>
</ol>
2. Antibody-Dependent Cellular Phagocytosis (ADCP) Assay Plate Preparation
<ol>
	<li>Conjugate biotinylated protein to 1 &#181;m microspheres (&#8216;beads&#39;; Table of Materials) according to the manufacturer&#39;s instructions.
	<ol>
		<li>Per plate of conjugated beads, incubate 6 &#181;g of protein with 12 &#181;L of stock beads in 200 &#181;L of 0.1% bovine serum albumin (BSA)-PBS at room temperature for 1 h, vortexing gently every 20 min.</li>
		<li>Centrifuge at 13,000 x g for 5 min. Discard supernatant, vortex gently and resuspend in 1200 &#181;L of 0.1% BSA-PBS. Repeat spin and wash step 2x. Resuspend in 1200 &#181;L of 0.1% BSA-PBS.</li>
	</ol>
	</li>
	<li>Aliquot 10 &#181;L of bead solution per well in a round bottom 96-well plate. Prepare dilutions of antibody or immune sera of interest in 12 &#181;L of 2% HSA HBSS, typically starting at 50 &#181;g/mL of antibody or a 1/100 serum dilution. 
	NOTE: In the sample data, monoclonal antibody (mAb) 830A was used.</li>
	<li>Add 10 &#181;L of titrated antibody/sera to the bead plate and incubate for 2 h at 37 &#176;C. Add 200 &#181;L of 2% HSA HBSS to each well and centrifuge plate at 2,000 x g for 10 min.</li>
	<li>Carefully remove supernatant by a rapid overturning and decanting of liquid from the plate wells into a sink to avoid disturbing the invisible bead pellet.</li>
</ol>
3. Breast milk cell isolation
<ol>
	<li>Obtain human breast milk from healthy lactating women, expressed using double electronic or manual pumps. Isolate cells within ~4 h of expression, keeping milk at room temperature.</li>
	<li>Using 50 mL tubes, centrifuge 50 mL milk at 800 x g for 15 min. Carefully pour off the skim milk and fat while leaving the cell pellet undisturbed. Wipe the inside of the tube with a lint-free wipe to remove all fat from the tube wall.</li>
	<li>Add 10 mL of 2% human serum albumen in Hank&#39;s balanced salt solution (2% HSA HBSS [without Ca2+ or Mg+]). Resuspend the pellet by gentle pipetting to avoid cell activation and apoptosis. Transfer to a 15 mL tube and centrifuge at 450 x g for 10 min.</li>
	<li>Pour off supernatant and repeat step 1.3. Then, gently resuspend cells in 1&#8722;2 mL of 2% HSA HBSS depending on expected cell number, and count cells by a hemocytometer or an automated cell counter, noting also the cell viability.</li>
</ol>
4. ADCP assay
NOTE: Methods described here are adapted from Ackerman et al.
<ol>
	<li>Add 50,000 freshly isolated BM cells to each ADCP assay plate well in 200 &#181;L of 2% HSA-HBSS. Incubate for 2 h at 37 &#176;C.
	<ol>
		<li>For control experiments, pre-incubate cells at 37 &#176;C with 10 &#181;g/mL actin inhibitor (cytochalasin-D), 50 &#181;g/mL Fc receptor blocking agent (FcBlock), or a combination of both prior to their addition to the plates.</li>
	</ol>
	</li>
	<li>Centrifuge plate at 930 x g for 10 min. Add 200 &#181;L of 2% HSA HBSS and repeat centrifugation. Carefully remove supernatant as in step 2.7 and repeat wash.</li>
	<li>Carefully remove supernatant and stain cells for viability using 0.5 &#181;g/mL (final concentration) fixable viability stain 450 per well in 50 &#181;L of 2% HSA HBSS. Incubate 20 min at room temperature in the dark. Centrifuge plate at 930 x g for 10 min and remove supernatant as in step 2.7. Add 200 &#181;L of 2% HSA HBSS and centrifuge plate again followed by removal of supernatant as in step 2.7.</li>
	<li>After viability staining, stain cells for leukocyte markers of interest, minimally including a CD45-specific stain such as PE-mouse anti-human CD45 (clone HI30) at an optimized concentration (1 &#181;g/&#181;L in 50 &#181;L of 1% BSA HBSS in the example data). 
	NOTE: Any lineage-specific markers of interest can be included.</li>
	<li>Incubate 20 min at room temperature in the dark. Centrifuge plate at 930 x g for 10 min and remove supernatant as in step 2.7. Add 200 &#181;L of 1% BSA HBSS and repeat centrifugation. Remove supernatant. Fix cells in 200 &#181;L of 0.5% formaldehyde in the dark at room temperature for 30 min or overnight at 4 &#176;C. Refrigerate in the dark until analysis.</li>
</ol>
5.&#160; Analysis by flow cytometry
<ol>
	<li>Perform initial gating to eliminate doublets on a forward scatter (FSC) vs. side scatter (SSC) plot and debris (material smaller than FSC = 5000) (see Figure 1). Use an SSC vs. viability stain (V450 in this case) plot to eliminate dead cells (those that are positive for viability stain).</li>
	<li>Use an SSC vs. CD45 plot to differentiate the major leukocyte classes (granulocytes, monocytes, lymphocytes) as extensively described. 
	NOTE: This classification is only suggestive and lineage-specific markers are needed to confirm cell type.</li>
	<li>For all CD45+ cells, or for each leukocyte subset of interest, measure ADCP activity by gating with a marker on the bead-positive cells in a histogram of the fluorescein isothiocyanate (FITC) channel, where the fluorescent beads are detected. 
	NOTE: The negative control wells in which beads were not added will indicate where the bead-positive cells are apparent in the histogram and therefore where to place the gating marker.</li>
	<li>Calculate ADCP scores as (median fluorescence intensity [MFI] of bead-positive cells) x (% of total CD45 + cells in the positive population). Use graphics software to plot scores at each Ab/serum concentration and to perform an area-under-the-curve (AUC) analysis. 
	NOTE: ADCP is considered positive if the AUC is greater than 3x standard deviation of the ADCP score AUC of a non-specific negative control mAb (in this case, 3865).</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 &#181;m FluoSpheres NeutrAvidin-Labeled Microspheres</td>
			<td>Thermo Fisher</td>
			<td>F8776</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Pharmingen PE Mouse Anti-Human CD45</td>
			<td>BD</td>
			<td>560975</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum Albumin</td>
			<td>MP Biomedicals</td>
			<td>8810025</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning V-bottom polystyrene 96-well plate</td>
			<td>Corning</td>
			<td>3894</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytochalasin D</td>
			<td>Sigma</td>
			<td>22144-77-0</td>
			<td></td>
		</tr>
		<tr>
			<td>EZ-Link NHS-LC-LC-Biotin kit</td>
			<td>Thermo Fisher</td>
			<td>21338</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 15mL Conical Centrifuge Tubes</td>
			<td>Corning</td>
			<td>352196</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50mL Conical Centrifuge Tubes</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixable Viability Stain 450</td>
			<td>BD</td>
			<td>562247</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde solution</td>
			<td>Sigma</td>
			<td>252549</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS without Calcium, Magnesium or Phenol Red</td>
			<td>Life Technologies</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Human BD Fc Block</td>
			<td>BD</td>
			<td>564219</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Serum Albumin</td>
			<td>MP Biomedicals</td>
			<td>2191349</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimtech Science Kimwipes Delicate Task Wipers</td>
			<td>Kimberly-Clark Professional</td>
			<td>34120</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1x pH 7.4</td>
			<td>Thermo Fisher</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene 10mL Serological Pipettes</td>
			<td>Corning</td>
			<td>4488</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein Concentrators PES, 3K MWCO, 0.5 mL</td>
			<td>Pierce</td>
			<td>88512</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Powell, R. L., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/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.

<img alt="Figure 1" src="/files/ftp_upload/22130/22130_Figure1.jpg" /> 
Figure 1: Sample flow cytometry data of cells isolated from breast milk. Cells were processed and stained as described in the protocol. (A) Single cells were gated on to eliminate doublets in an FSC-H vs. FSC-A plot as shown, also gating out the small debris &#60;5,000 in FSC-A. (B) This population was used to gate on live cells (which do not stain with the viability dye) in an SSC vs. V450 (viability stain) plot. (C) These live cells were used in an FSC vs. SSC plot. The expected position of non-leukocytes, likely to be predominantly mammary epithelial cells, is highlighted (&#34;E&#34;). (D) The same FSC vs. SSC plot is shown only with CD45+ cells. The major leukocyte subsets noted are only purported identities based on well-established and expected SSC parameters (G = granulocytes; M = monocytes; L = lymphocytes). (E) Viable cells were used for an SSC vs. CD45 plot with the major leukocyte subsets noted. Back-gating from this plot yielded the data shown in panel D. Note that this classification is only suggestive and that lineage-specific markers are needed to confirm cell type.

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

	...

Take fluorescently labeled microspheres conjugated to an antigen of interest. The immobilized antigens are bound to antigen-specific antibodies.
Add leukocytes, including non-phagocytic lymphocytes, phagocytic granulocytes, and monocytes. Incubate the mixture under physiological conditions.
The Fc region of the immobilized antibody binds to the Fc receptor on the phagocyte, triggering antibody-dependent cellular phagocytosis, or ADCP.
Centrifuge to pellet the cells and discard the supernatant containing non-internalized microspheres.
Add a viability stain that enters dead cells, fluorescently labeling them.
Centrifuge to pellet the cells and remove the supernatant.
Add fluorescently labeled antibodies that bind to leukocytes.
Centrifuge to discard the supernatant and fix the cells.
Using flow cytometry, first, eliminate the dead cells that are positive for viability stain.
Then, select the viable leukocytes and discriminate between granulocytes, monocytes, and lymphocytes based on their granularity and scattering properties.
Measure the fluorescence intensity of the microsphere-positive phagocytic cells to quantify their ADCP activity.

42 조회수. 식세포에 의한 항체 의존성 세포 식세포작용을 측정하기 위한 시험관 분석

비디오: 식세포에 의한 항체 의존성 세포 식세포작용을 측정하기 위한 시험관 분석 - 실험

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.

단핵구 단층 분석의 사용은 Fcγ 수용체 - 매개 식균 작용을 평가하기

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

연구

JoVE Journal

면역학 및 감염병학

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and inflammatory intractable diseases. We herein developed an experimental method to investigate phagocytosis quantitatively using the fruit fly Drosophila, in which the gene network controlling engulfment reactions is evolutionally conserved from mammals. In order to accurately detect and count engulfing and un-engulfing phagocytes using whole animals, Drosophila embryos were homogenized to obtain dispersed cells including phagocytes and apoptotic cells. The use of dispersed embryonic cells enables us to measure in vivo phagocytosis levels as if we performed an in vitro phagocytosis assay in which it is possible to observe all phagocytes and apoptotic cells in whole embryos and precisely quantify the level of phagocytosis. We confirmed that this method reproduces those of previous studies that identified the genes required for the phagocytosis of apoptotic cells. This method allows the engulfment of dead cells to be analyzed, and when combined with the powerful genetics of Drosophila, will reveal the complex phagocytic reactions comprised of the migration, recognition, engulfment, and degradation of apoptotic cells by phagocytes.

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

We herein describe a phagocytosis assay using the dispersed embryonic cells of Drosophila. It enables us to easily and precisely quantify in vivo phagocytosis levels, and to identify new molecules required for the phagocytosis of apoptotic cells.

에있는 Apoptotic 세포를위한 Phagocytosis 검정<em&gt; Drosophila</em&gt; 태아

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and ...

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are the main branches of innate immunity, and many of the factors regulating these responses are evolutionarily conserved between invertebrates and mammals. Phagocytosis, the central component of cellular innate immunity, is carried out by specialized blood cells of the immune system. The fruit fly, Drosophila melanogaster, has emerged as a powerful genetic model to investigate the molecular mechanisms and physiological impacts of phagocytosis in whole animals. Here we demonstrate an injection-based in vivo phagocytosis assay to quantify the particle uptake and destruction by Drosophila blood cells, hemocytes. The procedure allows researchers to precisely control the particle concentration and dose, making it possible to obtain highly reproducible results in a short amount of time. The experiment is quantitative, easy to perform, and can be applied to screen for host factors that influence pathogen recognition, uptake, and clearance.

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial infections.

과일 파리, 초파리 melanogaster에 비보 먹어서 분석 결과 사용 하 여의 세포 면역 반응 평가

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are ...

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

내레이션 대본

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