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). Virtually no studies have examined cells in human BM past the colostrum phase. Studies using colostral cells have generally concluded that the granulocytes in colostrum are less active than those isolated from blood, behaving as an &#39;exudate cell&#39; that has moved into the extravascular space35, though conflicting studies have reported similar phagocytic and bactericidal capacities36.
For decades, traditional microscopy was used to identify BM leukocytes, and this type of visual identification may have led to cell misidentification1. Few studies have compared BM leukocyte composition beyond the first month of lactation, and most have focused on colostrum. The use of flow cytometry to identify cells is likely to accurately identify cells, though only a small number of BM studies have been done using this method, often with a very small sample number. Current studies have indicated that the leukocyte content of BM at all stages of lactation varies widely, ranging from ~104&#8722;7 x 105 leukocytes/mL in early colostrum, decreasing to 103&#8722;5 x 104 leukocytes/mL in mature milk, though all studies confirm that cell concentration and composition is impacted strongly by the stage of lactation1,2,3,4,5. As milk transitions to its mature composition, neutrophil concentration appears to increase, though such studies have not typically extended beyond the first month postpartum34.
The protocol described herein uses fluorescent beads as the phagocytic target, though it likely can be applied to study BM ADCP of a variety of more biologically relevant targets such as immune complexes and infected cells, triggered by various Ab isotypes and subclasses. A larger cell staining panel can be employed to further differentiate the leukocytes. Large studies will be essential to develop a comprehensive understanding of ADCP by these relevant primary cells. This protocol allows for the establishment of ADCP by BM leukocytes as a potential mechanism for reduction of MTCT of HIV, as well as other pathogens.

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.

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			<td height="21" style="height:21px;width:356px;">1 &#181;m FluoSpheres NeutrAvidin-Labeled Microspheres&#160;</td>
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			<td height="21" style="height:21px;">Corning V-bottom polystyrene 96-well plate&#160;</td>
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			<td>22144-77-0</td>
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			<td height="21" style="height:21px;">EZ-Link NHS-LC-LC-Biotin kit&#160;</td>
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			<td height="21" style="height:21px;width:356px;">Falcon 15mL Conical Centrifuge Tubes</td>
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			<td style="width:85px;">352196</td>
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			<td height="21" style="height:21px;width:356px;">Falcon 50mL Conical Centrifuge Tubes</td>
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			<td>352070</td>
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			<td height="21" style="height:21px;">Formaldehyde solution</td>
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			<td>252549&#160;</td>
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			<td height="21" style="height:21px;width:356px;">HBSS without Calcium, Magnesium or Phenol Red</td>
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			<td style="width:85px;">14175-095</td>
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			<td height="21" style="height:21px;">Human BD Fc Block</td>
			<td>BD</td>
			<td style="width:85px;">564219</td>
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			<td height="21" style="height:21px;">Human Serum Albumin</td>
			<td style="width:193px;">MP Biomedicals</td>
			<td style="width:85px;">2191349</td>
			<td></td>
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			<td height="21" style="height:21px;width:356px;">Kimtech Science Kimwipes Delicate Task Wipers</td>
			<td>Kimberly-Clark Professional&#160;</td>
			<td>34120</td>
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			<td height="21" style="height:21px;width:356px;">PBS 1x pH 7.4</td>
			<td style="width:193px;">Thermo Fisher</td>
			<td style="width:85px;">10010023</td>
			<td></td>
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			<td height="21" style="height:21px;width:356px;">Polystyrene 10mL Serological Pipettes&#160;</td>
			<td style="width:193px;">Corning&#160;</td>
			<td>4488</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Protein Concentrators PES, 3K MWCO, 0.5 mL</td>
			<td style="width:193px;">Pierce</td>
			<td style="width:85px;">88512</td>
			<td></td>
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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;222,857 cells/mL. Figure 1 illustrates the gating strategy eliminating doublets, debris, and dead cells. Viability was ~90&#8722;99%. Approximately 1.6&#8722;12.3% of total live cells were CD45+ leukocytes (Table 1). Most purported monocytes appeared to be precursors/immature cells as previously described, based on the suggestive SSC vs. CD45 gating34. The purported monocytes were defined as SSClow-intermediate/CD45low, though few exhibited the higher CD45 staining levels distinct from the purported lymphocyte population (SSClow/CD45low) more typically associated with blood monocytes (Figure 1), similar to previous studies33,34. The purported granulocytes were defined as SSChigh/CD45intermediate33,34 (Table 1). Note that this classification is only suggestive and that lineage-specific markers would be needed to confirm cell type.
ADCP activity of the freshly isolated BM cells was measured using the HIV-specific human mAb 830A, which is specific for the V2 region of the HIV envelope and binds to the V1V2-2F5K antigen tested here. ADCP activity was measured for the example here using milk obtained at 1 month post-partum (Figure 2A). Example data shows the expected FITC (bead+) histograms seen when gating on CD45+ cells (data generated using 1 &#181;g/mL mAb is shown). The black markers indicate the populations used to calculate ADCP scores. In the sample 830A data (first panel of Figure 2A), percentage of CD45+ cells and mean fluorescence of that population are shown, which were used to calculate the ADCP score using the equation in step 3.4. Cells pre-incubated with actin inhibitor cytochalasin-D (cytoD) and/or FcR-blocking Abs (FcBlock) prior to their incubation with the Ab-bound/antigen-coupled beads exhibited ADCP activity at the level of the control mAb 3865 or below, indicating ADCP was FcR and actin-dependent (Figure 2). The ADCP score determined for total CD45+ cells was ~25&#8722;35-fold above background levels defined using the negative control anti-anthrax mAb 3865. Each major subset was analyzed separately as well. The purported granulocytes exhibited ADCP activity ~12&#8722;29-fold higher than background. The purported monocyte ADCP was ~2&#8722;3-fold above background (Figure 2). The purported lymphocytes as expected did not exhibit any measurable ADCP activity (less than 3x standard deviation of the ADCP score AUC of the non-specific negative control mAb 3865; data not shown).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60149/60149fig1.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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60149/60149fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60149/60149fig2.jpg" /> 
Figure 2: Sample ADCP data using cells isolated from breast milk. The ADCP assay performed is based on the assay adapted from Ackerman et al.30. The assay was performed as outlined in the protocol above. (A) Sample FITC histograms at 1 &#181;g/mL mAb, with markers indicating the bead/FITC+ populations used to determine the ADCP score. Scores were calculated as (MFI of bead-positive cells) x (% of total CD45+ cells in the bead/FITC+ positive population). The first panel using mAb 830A alone also indicates the percentage of total CD45+ cells and the mean fluorescence intensity value used to calculate the ADCP score in that example. (B) ADCP scores at each mAb dilution assayed were used to calculate area-under-the-curve (AUC) values in graphics software. For control experiments, actin inhibitor cytochalasin-D (cytoD), FcR blocking agent (FcBlock), or a combination of both were pre-incubated with cells prior to their addition to the immune complexes (see legends). Note that this cell classification is only suggestive and that lineage-specific markers are needed to confirm cell type. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60149/60149fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample</td>
			<td>Cells/mL</td>
			<td>% CD45+</td>
			<td>% Granulocytes*&#160;</td>
			<td>% Monocytes*</td>
		</tr>
		<tr>
			<td>1</td>
			<td>222,857</td>
			<td>12.3 &#177; 1.9</td>
			<td>13.6 &#177; 3.8</td>
			<td>65.9 &#177; 5.6</td>
		</tr>
		<tr>
			<td>2</td>
			<td>27,361</td>
			<td>1.6 &#177; 0.01</td>
			<td>25.2 &#177; 4.0</td>
			<td>9.1 &#177; 5.6</td>
		</tr>
		<tr>
			<td>3</td>
			<td>161,486</td>
			<td>3.6 &#177; 1.1</td>
			<td>47.8 &#177; 6.8</td>
			<td>24.3 &#177; 4.3</td>
		</tr>
		<tr>
			<td>4</td>
			<td>16,083</td>
			<td>2.7 &#177; 0.1</td>
			<td>17.9 &#177; 3.5</td>
			<td>34.4 &#177; 1.0</td>
		</tr>
		<tr>
			<td>5</td>
			<td>25,155</td>
			<td>4.0 &#177; 0.7</td>
			<td>29.7 &#177; 2.6</td>
			<td>20.5 &#177; 1.4</td>
		</tr>
		<tr>
			<td colspan="2">*of CD45+ cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Examples of typical breast milk cell counts and characteristics.

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

7.1K Views.  Icahn School of Medicine at Mount Sinai. Breast milk transmits human immunodeficiency virus (HIV), though only ~15% of infants breastfed by HIV-infected mothers become infected. Breastfed infants ingest ~105−108 maternal leukocytes daily, though these cells are understudied. Here we describe the isolation of breast milk leukocytes and an analysis of their phagocytic capacity.

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

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Isolation of Brain-infiltrating Leukocytes

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine encephalomyelitis virus (TMEV) via a rapid intracranial injection technique and how to purify a leukocyte-enriched population of infiltrating cells from whole brain. Briefly, mice are anesthetized with isoflurane in a closed chamber and are free-hand injected with a Hamilton syringe into the frontal cortex. Mice are then killed at various times after infection by isoflurane overdose and whole brains are extracted and homogenized in RPMI with a Tenbroeck tissue grinder. Brain homogenates are centrifuged through a continuous 30% Percoll gradient to remove the myelin and other cell debris. The cell suspension is then strained at 40 &mu;m, washed and centrifuged on a discontinuous Ficoll-Paque Plus gradient to select and purify the leukocytes. The leukocytes are then washed and resuspended in appropriate buffers for immunophenotyping by flow cytometry. Flow cytometry reveals a population of innate immune cells at the early stages of infection in C57BL/6 mice. At 24 hours post infection, multiple subsets of immune cells are present in the BILs, with an enriched population of Gr1+, CD11b+ and F4/80+cells. Therefore, this method is useful in characterizing the immune response to acute infection in the brain.

A rapid method to obtain infiltrating leukocytes from the murine brain is described. This method utilizes a continuous Percoll gradient and discontinuous Ficoll gradient to select and purify the leukocyte-enriched layer. Isolated leukocytes may then be characterized by flow cytometric measurements.

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

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 Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

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. ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

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 ...

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.

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

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Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

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

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos