We thank Dr. Susan Zolla-Pazner in the Department of Medicine and Department of Microbiology at the Icahn School of Medicine at Mount Sinai for manuscript review. The NIH/NICHD provided funding for this project under grant number R21 HD095772-01A1. In addition, R. Powell was supported by funds from the Department of Medicine, Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai.

Each participant in this study was recruited and interviewed in accord with the ethical and institutional review board (IRB) approval with the guidance and authorization of Mount Sinai&#8217;s Program for the Protection of Human Subjects (PPHS) using an IRB-approved protocol for obtaining breast milk samples.
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 envelope30.</li>
	<li>Perform biotinylation using a commercial kit (Table of Materials) according to the manufacturer&#8217;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)30,31. 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&#8217;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&#8217;; Table of Materials) according to the manufacturer&#8217;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&#160;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&#160;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&#8217;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.32.
<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 FcR 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 described33,34. 
	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>

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The authors have nothing to disclose.

The flow cytometry-based technique for measuring ADCP activity described herein was first described in 201130 and has since been proven robust and cited in more than 80 studies. The protocol described here adapts this technique for use with primary BM cells for the first time. Previous studies of Fc-mediated functionality by BM cells have been largely limited to measurement of oxidative bursts or histology-based phagocytosis assays using cells isolated from colostrum (0&#8722;4 days after birth). ...

Human breast milk (BM) is comprised of maternal cells that are &#62;90% viable1. Cell composition is impacted strongly by stage of lactation, health status of mother and infant, and individual variation, which remains poorly understood1,2,3,4. Given that BM contains ~103&#8722;105 leukocytes/mL, it can be estimated that breastfed infants ingest ~105&#8722;108 maternal leukocytes daily5. Various in vivo studies have demonstrated that maternal leukocytes provide critical immunity to the infant and are functional well beyond these sites of initial ingestion5,6,7,8,9,10,11. All maternally-derived cells ingested by the infant have the potential to perform immune functions alongside or to compensate for the infant&#39;s own leukocytes12.
Mother-to-child transmission (MTCT) of human immunodeficiency virus (HIV) remains a crisis in resource-limited countries. As diarrheal and respiratory diseases are responsible for substantial rates of mortality among infants in resource-limited countries, and these illnesses are significantly reduced by exclusive breastfeeding, the benefits to HIV-infected mothers of breastfeeding far outweigh the risks13,14,15. Unless access to clean water and appropriate infant formula is reliable, the WHO does not recommend cessation of breastfeeding for HIV-infected mothers16. Approximately 100,000 MTCTs via BM occur annually; yet, only ~15% of infants breastfed by their HIV-infected mothers become infected, suggesting a strong protective effect of BM17,18,19,20,21. Numerous factors likely work in tandem to prevent transmission. Importantly, HIV-specific antibodies (Abs) in BM have been correlated with reduced MTCT and/or reduced infant death from HIV infection22,23. What remains largely unclear is the contribution of the cellular fraction of BM to its antiviral qualities.
Many Abs facilitate a variety of anti-viral activities mediated by the &#39;constant&#39; region of the immunoglobulin molecule, the crystallizable fragment (Fc), via interaction with Fc receptors (FcRs) found on virtually all innate immune cells, virtually all of which are found in human BM24. Antibody-dependent cellular phagocytosis (ADCP) has been demonstrated as necessary for the clearance of viral infections and has been understudied in the case of prevention of MTCT of HIV25,26,27,28,29. Given the paucity of knowledge about the potential contribution of ADCP activity by BM phagocytes to prevention of MTCT of HIV, we aimed to develop a rigorous method to isolate cells from human BM in order to undertake a study of ADCP mediated by cells from BM obtained at various stages of lactation.

<table><tbody><tr><td>1 &amp;micro;m FluoSpheres NeutrAvidin-Labeled Microspheres&amp;nbsp;</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&amp;nbsp;</td><td>Corning&amp;nbsp;</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&amp;nbsp;</td><td>Thermo Fisher</td><td>21338</td><td></td></tr><tr><td>Falcon 15 mL Conical Centrifuge Tubes</td><td>Corning&amp;nbsp;</td><td>352196</td><td></td></tr><tr><td>Falcon 50 mL Conical Centrifuge Tubes</td><td>Corning&amp;nbsp;</td><td>352070</td><td></td></tr><tr><td>Fixable Viability Stain 450&amp;nbsp;</td><td>BD</td><td>562247</td><td></td></tr><tr><td>Formaldehyde solution</td><td>Sigma</td><td>252549&amp;nbsp;</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&amp;nbsp;</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 10 mL Serological Pipettes&amp;nbsp;</td><td>Corning&amp;nbsp;</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>

isolation of leukocytes from human breast milk for use in an antibody-dependent cellular phagocytosis assay of hiv targets

Even in the absence of antiretroviral drugs, only ~15% of infants breastfed by HIV-infected mothers become infected, suggesting a strong protective effect of breast milk (BM). Unless access to clean water and appropriate infant formula is reliable, the WHO does not recommend cessation of breastfeeding for HIV-infected mothers. Numerous factors likely work in tandem to reduce BM transmission. Breastfed infants ingest ~105&#8722;108 maternal leukocytes daily, though what remains largely unclear is the contribution of these cells to the antiviral qualities of BM. Presently we aimed to isolate cells from human BM in order to measure antibody-dependent cellular phagocytosis (ADCP), one of the most essential and pervasive innate immune responses, by BM phagocytes against HIV targets. Cells were isolated from 5 human BM samples obtained at various stages of lactation. Isolation was carried out via gentle centrifugation followed by careful removal of milk fat and repeated washing of the cell pellet. Fluorescent beads coated with HIV envelope (Env) epitope were used as targets for analysis of ADCP. Cells were stained with the CD45 surface marker to identify leukocytes. It was found that ADCP activity was significant above control experiments and reproducibly measurable using an HIV-specific antibody 830A.

Milk can be kept at room temperature or cooler (though not frozen); however, given that we have observed reduced viability when milk has been kept very cold (data not shown), and that it is simpler to collect, store briefly, and transport at ambient temperatures, it is recommended that samples are not refrigerated in order to reduce sample-to-sample variability. In milk obtained 7&#8722;183 days post-partum, cell concentration determined by automated cell counter ranged from 16,083&#8722;...

Watch this Scientific Journal Video about Isolation of Leukocytes from Human Breast Milk for Use in an Antibody-dependent Cellular Phagocytosis Assay of HIV Targets at JoVE.com

Isolation of Leukocytes from Human Breast Milk for Use in an Antibody-dependent Cellular Phagocytosis Assay of HIV Targets

Breast milk transmits human immunodeficiency virus (HIV), though only ~15% of infants breastfed by HIV-infected mothers become infected. Breastfed infants ingest ~105&#8722;108 maternal leukocytes daily, though these cells are understudied. Here we describe the isolation of breast milk leukocytes and an analysis of their phagocytic capacity.

Even in the absence of antiretroviral drugs, only ~15% of infants breastfed by HIV-infected mothers become infected, suggesting a strong protective effect ...

isolation-leukocytes-from-human-breast-milk-for-use-an-antibody

Research

JoVE Journal

Immunology and Infection

7.1K Views.  Icahn School of Medicine at Mount Sinai. We ontwikkelden een rigoureuze methode voor het isoleren van cellen uit menselijke moedermelk om antilichaamafhankelijke cellulaire fagocytose, of ADCP, te bestuderen door borstkankerfgocyten tegen HIV-doelen. Deze techniek vergemakkelijkt de relatief snelle en eenvoudige isolatie van moedermelkcellen om de capaciteit van leukocytenpopulaties om ADCP uit te voeren te evalueren. Het aantonen van de procedure zal Alisa Fox zijn, een technicus van mijn laboratorium.Na het selecteren van e...

Video: Isolation of Leukocytes from Human Breast Milk for Use in an Antibody-dependent Cellular Phagocytosis Assay of HIV Targets

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.
Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.
To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are used for host defense. For the quantitative determination of the functional status of the humoral immune response of an individual, both serum Abs and circulating ASCs are commonly measured as functional readouts. In humans, peripheral blood is the most convenient and readily accessible sample that can be used for the determination of the humoral immune response elicited by host B cells. Distinct B-cell subsets, including ASCs, can be isolated directly from peripheral blood via selection with lineage-specific Ab-conjugated microbeads or via cell sorting with flow cytometry. Moreover, purified na&#239;ve and memory B cells can be activated and differentiated into ASCs in culture. The functional activities of ASCs to contribute to Ab secretion can be quantified by ELISpot, which is an assay that converges enzyme-linked immunoabsorbance assay (ELISA) and western blotting technologies to enable the enumeration of individual ASCs at the single-cell level. In practice, the ELISpot assay has been increasingly used to evaluate vaccine efficacy because of the ease of handling of a large number of blood samples. The methods of isolating human B cells from peripheral blood, the differentiation of B cells into ASCs in vitro, and the employment of ELISpot for the quantification of total IgM- and IgG-ASCs will be described here.

Human peripheral blood is commonly used for the assessment of the humoral immune response. Here, the methods for isolating human B cells from peripheral blood, differentiating human B cells into antibody (Ab)-secreting B cells (ASCs) in culture, and enumerating the total IgM- and IgG-ASCs via an ELISpot assay are described.

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

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 ...

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment

Bronchoscopy is a medical procedure whereby normal saline is injected into the lungs via a bronchoscope and then suction is applied, removing bronchoalveolar lavage (BAL) fluid. The BAL fluid is rich in cells and can thus provide a 'snapshot' of the pulmonary immune milieu. CD4 T cells are the best characterized HIV reservoirs, while there is strong evidence to suggest that tissue macrophages, including alveolar macrophages (AMs), also serve as viral reservoirs. However, much is still unknown about the role of AMs in the context of HIV reservoir establishment and maintenance. Therefore, developing a protocol for processing BAL fluid to obtain cells that may be used in virological and immunological assays to characterize and evaluate the cell populations and subsets within the lung is relevant for understanding the role of the lungs as HIV reservoirs. Herein, we describe such a protocol, employing standard techniques such as simple centrifugation and flow cytometry. The CD4 T cells and AMs may then be used for subsequent applications, including immunophenotyping and HIV DNA and RNA quantification.

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment

We describe a method for processing bronchoalveolar lavage fluid and matched peripheral blood from chronically HIV-infected individuals on antiretroviral therapy to assess pulmonary HIV reservoirs. These methods result in the acquisition of highly pure CD4 T cells and alveolar macrophages that may subsequently be used for immunophenotyping and HIV DNA/RNA quantifications by ultrasensitive polymerase chain reaction.

Bronchoscopy is a medical procedure whereby normal saline is injected into the lungs via a bronchoscope and then suction is applied, removing ...

Isolation, Transfection, and Culture of Primary Human Monocytes

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. While antiretroviral therapy efficiently lowers systemic viral load and restores normal CD4+ T cell counts, it does not reconstitute a completely functional immune system. A dysfunctional immune system in HIV-infected individuals undergoing cART may be characterized by immune activation, early aging of immune cells, or persistent inflammation. These conditions, along with comorbid factors associated with HIV infection, add complexity to the disease, which cannot be easily reproduced in cellular and animal models. To investigate the molecular events underlying immune dysfunction in these patients, a system to culture and manipulate human primary monocytes in vitro is presented here. Specifically, the protocol allows for the culture and transfection of primary CD14+ monocytes obtained from HIV-infected individuals undergoing cART as well as from HIV-negative controls. The method involves isolation, culture, and transfection of monocytes and monocyte-derived macrophages. While commercially available kits and reagents are employed, the protocol provides important tips and optimized conditions for successful adherence and transfection of monocytes with miRNA mimics and inhibitors as well as with siRNAs.

Presented here is an optimized protocol for isolating, culturing, transfecting, and differentiating human primary monocytes from HIV-infected individuals and healthy controls.

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. ...

High-Throughput PBMC Isolation Using Immunomagnetic Beads

Isolating Human Peripheral Blood Mononuclear Cells from Buffy Coats via High Throughput Immunomagnetic Bead Separation

Peripheral blood mononuclear cells (PBMCs) are a heterogeneous population of monocytes and lymphocytes. Cryopreserved PBMCs have stable viability in long-term storage, making them an ideal cell type for many downstream research purposes, including flow cytometry, immunoassays, and genome sequencing. Typically, PBMCs are isolated via&#160;density gradient centrifugation, however, it is a low-throughput workflow that is difficult and costly to scale. This article presents a high-throughput workflow using a magnetic bead-based PBMC isolation method that is quick to implement. Total cell concentration, viability, and population distribution with PBMCs obtained using density gradient isolation were compared, and cell viability and proportion of cell types were comparable for both techniques. Isolated PBMCs demonstrated over 70% viability up to 9 days after blood collection, although yield decreased by half after 5 days compared to PBMCs processed within 24 h of collection. In summary, this article describes a PBMC protocol that utilizes a bead-based approach to adapt to a high throughput workflow and demonstrates that both manual and automated bead-based methods can increase processing capacity and provide flexibility for various budgets.

Author Spotlight: Advancements in PBMC Isolation &#8211; Enhancing Throughput and Consistency in Research

This protocol details a high-throughput automation-compatible method to isolate human peripheral blood mononuclear cells for biobanking and other purposes.

Peripheral blood mononuclear cells (PBMCs) are a heterogeneous population of monocytes and lymphocytes. Cryopreserved PBMCs have stable viability in ...

Biology

Isolation of Monocytes from Whole Blood by Immunomagnetic Negative Selection

Source: Plaisance-Bonstaff, K., et al. <a href="https://app.jove.com/v/59967">Isolation, Transfection, and Culture of Primary Human Monocytes.</a> J. Vis. Exp. (2019)
In this video, we demonstrate the isolation of monocytes from whole human blood by immunomagnetic negative separation. The isolated monocytes can be used for further downstream analysis.

Source: Plaisance-Bonstaff, K., et al. Isolation, Transfection, and Culture of Primary Human Monocytes. J. Vis. Exp. (2019)
In this video, we demonstrate ...

JoVE Encyclopedia of Experiments

Immunology

Immune Systems and Components

Cell Isolation and Culture

Isolation and Purification of B Cells from Human Peripheral Blood

Source: Tzeng, S. J. <a href="https://app.jove.com/v/54582">The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity.</a> J. Vis. Exp. (2016)
In this video, we demonstrate the isolation of B cells from human peripheral blood using biotinylated antibodies specific for non-B cells and streptavidin-conjugated magnetic microbeads.

Source: Tzeng, S. J. The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of ...

Isolation of Leukocytes from Human Breast Milk for Use in an Antibody-dependent Cellular Phagocytosis Assay of HIV Targets

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