We thank Hannah Puzas for assistance with system design and configuration, and Kevin Khovananth for technical advice. Funding for this work was provided by The Hearst Foundations and the Providence Portland Medical Foundation.

<ol>
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YK, ILG, TLM, WLM, DPH, and KSB: No competing financial interests. ANT is an employee of Beckman Coulter, Inc. Publication of this video article is sponsored by Beckman Coulter, Inc.

Immunophenotyping of peripheral blood is critically important for gaining insights into individual responses to immunotherapy. The challenge lies in assay standardization to control experiment-to-experiment variability1,14. One main source of variability lies in the human manipulation of samples. Therefore, it is conceivable that partial or full automation of sample processing will facilitate a dramatic reduction in experiment-to-experiment variability1,14. In this protocol, we report our successful effort to minimize assay variability by introducing an automated liquid handler equipped with 2D barcode reader for sample staining in whole blood immunophenotyping.
In design, our method is versatile and can monitor other immune subsets by adding additional &#34;Base Cocktails&#34; to define them. Such subsets include T helper cells, T regulatory cells, B cells, NK cells, dendritic cells, and monocytes (manuscript in preparation)18. We adapted the consortium&#39;s recommended antibody panels by introducing additional markers1. Cell populations were identified using &#34;Base Cocktail&#34; conjugated to fluorochromes with minimal emission into the brilliant violet 421 (BV421), phycoerythrin (PE), and allophycocyanin (APC) detectors, as we wanted to reserve these channels for the detection of inducible antigens. This feature not only ensures the highest sensitivity to monitor activation status of immune cells but also enables flexible accommodation of new activation markers as these three fluorochromes are most frequently conjugated with antibodies against inducible markers. Therefore, our method is not only suitable for cocktail preparation for whole blood immunophenotyping but is also useful for staining other samples including peripheral blood mononuclear cells and cells recovered from disaggregated tissues (e.g., tumor).
Successful introduction of our method requires special attention to steps in labware preparation, and programing and execution of the method. Preparation of the 2D barcode tubes includes labeling with human-readable labels and transferring of the antibodies to the designated tubes. To prevent the introduction of errors, we recommend doing this with two people. Whenever changing spreadsheets, investigators should verify whether the method works properly. Choosing appropriate pipetting methods during programing ensures successful liquid transfer. LLS enables pipetting without getting precipitated debris from the bottom of antibody tubes, for which we use either P50 or P200 conductive tips. LLS may not be a good option for P1000 tips as LLS is more sensitive with P1000 tips and sometimes falsely triggered by presence of bubbles. Heights in labware definition should be adjusted for each instrument as suboptimal liquid transfer could occur, for example, if there is not enough space between the edge of tips and the bottom of tubes. As shown in Figure 12, if the &#34;Base Cocktail&#34; and/or &#34;Activation Cocktail&#34; are not vortexed well, it may result in suboptimal staining.
We thought about using lyophilized reagents for cell staining as an alternative approach to reduce variability related with reagent dispensing19. However, polymer conjugates of brilliant violet dyes are known to interact with each other causing non-specific signals. As such, adding more than two polymer conjugates (e.g., BV421 and BV650 etc.) in lyophilized reagents may cause non-specific signaling (e.g., increase of BV650 background signal in BV421+ population)20. Moreover, lyophilized reagents lack flexibility for including new staining. They are usually more expensive and require a bulk order. For those reasons, we chose to use the automated liquid handler equipped with the 2D barcode tubes. Although it takes time to set up and entails an upfront investment to purchase the instrument, in the long run such factors will be compensated for by the increased productivity and reproducibility of assays. In fact, a few groups previously reported the successful integration of the automated liquid handler into their workflow of immunophenotyping or similar applications21,22. Automated solutions for flow cytometry analysis are also available from commercial sources (FACS SPA III, Automated Cocktail Preparation Workstation, and FlowStainer). This further indicates there is a great need for automated cocktail preparation for immunophenotyping.
After mastering this technique, we envision that the development of fully automated whole blood immunophenotyping will further reduce experiment-to-experiment variability and might make whole blood immunophenotyping feasible even in a multicenter clinical trial setting23. We have already begun using a lyse wash assistant to automate the lysis and washing steps. We also envision that automated determination of the volume of antibody in the 2D barcode tubes and the tracking of reagent dispensing will greatly improve inventory of antibodies and the quality control over our method, respectively.

Immunophenotyping of human peripheral blood determines quantitative and qualitative changes in immune cell subsets1. It provides insight into mechanisms of action and resistance, aids discovery of predictive biomarkers, and facilitates the development of combination immunotherapies. Therefore, the validation and standardization of immunophenotyping is an area of considerable interest to academic researchers, clinical laboratories, and industries.
Although immunophenotyping of peripheral blood by flow cytometric methods is currently used for clinical management of hematological malignancies2,3 and human immunodeficiency virus (HIV) infection4,5, reliable immunophenotyping of peripheral blood for immunotherapy demands specific considerations in the validation and standardization because it requires more extensive coverage over immune cell subsets and activation/inhibitory receptors1,6-8. Successful immunophenotyping requires careful standardization to minimize experiment-to-experiment variability related to instrument setup and the cell staining procedure9,10. While standardization of instrument settings for flow cytometry is well established to address the former concern9,11,12, it remains unclear how to minimize variability related to cell staining without restricting coverage of immune cell subsets and their activation status.
As the number of antigens to be detected increases, so too does the opportunity for error and variability owing to suboptimal reagent dispensing and cross-contamination. Methods for the phenoptypic analysis of anticoagulated whole blood were established in the late 1980&#39;s for use in clinical laboratory assays. These same methods serve the needs of the research laboratory for sample preparation. Importantly, the antibody cocktails used in the clinical laboratory are typically less complex and available pre-titered and pre-mixed from the manufacturer. Only a single transfer of the pre-mixed antibody cocktail is required. In the research setting, antibody cocktails of 10-16 antibodies are typical. Each cocktail must be validated for stability by the laboratory or prepared fresh before each assay. Preparing multiple antibody cocktails for a sample might entail the pipetting of 50-80 individual antibodies, a task that is both tedious and error prone.
Automation of cocktail preparation has several advantages over manual cocktail preparation such as fewer errors, increased accuracy of pipetting, and possibly even decreased reagent wastage. Here, we report the successful introduction of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into the cell-staining process of immunophenotyping to minimize variability related to sample preparation.

<table border="0" cellpadding="0" cellspacing="0" width="1546">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:613px;">BD LSRFortessa</td>
			<td style="width:188px;">BD Biosciences</td>
			<td style="width:116px;"></td>
			<td style="width:629px;">Flow cytometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NXp Span-8 Laboratory Automation Workstation&#160;</td>
			<td>Beckman Coulter</td>
			<td>A31839</td>
			<td>Laboratory Automation Workstation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insert, Tube, 11 mm, White, for 1.5 mL Microfuge Tubes (case of 25)</td>
			<td>Beckman Coulter</td>
			<td>373696</td>
			<td>Accessary for 24-Position Tube Rack to accommodate microfuge tubes.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LLS kit</td>
			<td>Beckman Coulter</td>
			<td>719262</td>
			<td>Attach bottom of the deck in Laboratory Automation Workstation to enable liquid level sensing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-Position Tube Rack</td>
			<td>Beckman Coulter</td>
			<td>373661</td>
			<td>Tube rack to hold microfuge tubes for antibody cocktails or blood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BIOHAZARD AUTOCLAVE BAGS 12x24 IN. RED</td>
			<td>LabMart</td>
			<td>M111416</td>
			<td>Disposable autoclave bags for the Laboratory Automation Workstation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VisionMate High Speed 2D Barcode Reader</td>
			<td>Thermo Scientific</td>
			<td>AB-1850</td>
			<td>2D barcode reader</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">8-Channel Handheld Screw Cap Capper/Decapper</td>
			<td>Thermo Scientific</td>
			<td>4105MAT</td>
			<td>For de-capping and capping 2D barcode tubes&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>02-681-335</td>
			<td>For making cocktail in or supplying blood speciment to the Laboratory Automation Workstation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CoolRack XT CFT24</td>
			<td>biocision</td>
			<td>BCS-534</td>
			<td>To hold sample tubes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.0 mL Matrix ScrewTop Amber Tubes in Latch Racks</td>
			<td>Thermo Scientific</td>
			<td>3741AMB</td>
			<td>2D barcode tubes to store fluorescent dye-cojugated antibodies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biomek Span-8 P1000 Tips, Conductive, Pre-sterile with Barrier, 1025 &#181;L</td>
			<td>Beckman Coulter</td>
			<td>987925</td>
			<td>Tips for Laboratory Automation Workstation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biomek Span-8 P50 Tips, Conductive, Pre-sterile with Barrier, 50 &#181;L (Case of 10 racks)</td>
			<td>Beckman Coulter</td>
			<td>B01091</td>
			<td>Tips for Laboratory Automation Workstation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biomek Span-8 P250 Tips, Conductive, Pre-sterile with Barrier (Case of 10 racks)</td>
			<td>Beckman Coulter</td>
			<td>394627</td>
			<td>Tips for Laboratory Automation Workstation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD FACS Lysing Solution 10X Concentrate</td>
			<td>BD Biosciences</td>
			<td>349202</td>
			<td>RBC lysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Round-Bottom Polystyrene Tubes</td>
			<td>Fisher Scientific</td>
			<td>14-959-5</td>
			<td>Sample tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD45 FITC</td>
			<td>BD Biosciences</td>
			<td>347463</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD3 Alexa Fluor 700</td>
			<td>eBioscience</td>
			<td>56-0038-42</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD4 PerCPCy5.5</td>
			<td>BD Biosciences</td>
			<td>341654</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-TCRgd BV650</td>
			<td>BD Biosciences</td>
			<td>564156</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-TCRab BV786</td>
			<td>BD Biosciences</td>
			<td>563825</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD8 APC-H7</td>
			<td>BD Biosciences</td>
			<td>560179</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CCR7 PE-CF594</td>
			<td>BD Biosciences</td>
			<td>562381</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD45RA PE-Cy7</td>
			<td>BD Biosciences</td>
			<td>337167</td>
			<td>Effector T cell panel (base)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-HLA-DR BV421</td>
			<td>BD Biosciences</td>
			<td style="width:116px;">562804</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD278(ICOS) PE</td>
			<td>BD Biosciences</td>
			<td style="width:116px;">557802</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD38 APC</td>
			<td>BD Biosciences</td>
			<td style="width:116px;">340439</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD279(PD1) BV421</td>
			<td>BD Biosciences</td>
			<td style="width:116px;">562516</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD357(GITR) PE</td>
			<td>eBioscience</td>
			<td style="width:116px;">12-5875-42</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD137(41BB) APC</td>
			<td>BD Biosciences</td>
			<td style="width:116px;">550890</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD69 BV421</td>
			<td>BD Biosciences</td>
			<td style="width:116px;">562884</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD314(NKG2D) PE</td>
			<td>BD Biosciences</td>
			<td style="width:116px;">557940</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CD134(OX40) APC</td>
			<td style="width:188px;">BioLegend</td>
			<td>350008</td>
			<td>Effector T cell panel (activation)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Vacutainer Plastic Blood Collection Tubes with Sodium Heparin: Conventional Stopper</td>
			<td>Fisher Scientific</td>
			<td>02-689-6</td>
			<td>For collecting blood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumin, Bovine Serum, Fraction V, RIA and ELISA Grade</td>
			<td>EMD Millipore</td>
			<td>126593</td>
			<td>For flow wash buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Azide</td>
			<td>Sigma-Aldrich</td>
			<td>S8032</td>
			<td>For flow wash buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin, sodium salt</td>
			<td>Affimetrix</td>
			<td>16920</td>
			<td>For flow wash buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hank&#8217;s Balanced Salt Solution (HBSS), 1X, with Calcium, Magnesium, without Phenol Red</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30588.02</td>
			<td>For flow wash buffer</td>
		</tr>
	</tbody>
</table>

a semi-automated approach to preparing antibody cocktails for immunophenotypic analysis of human peripheral blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Watch this Scientific Journal Video about A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood at JoVE.com

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

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Research

JoVE Journal

Immunology and Infection

10.7K Views.  Providence Cancer Center, Providence Portland Medical Center. Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

Video: A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and infections using cost effective, high-throughput approaches would be indispensable. This manuscript describes the development and analysis of a bead-based multiplex immunoassay capable of measuring the presence of antibodies in human saliva to multiple pathogens simultaneously. Saliva is particularly attractive in this application because it is noninvasive, cheaper and easier to collect than serum. Antigens from environmental pathogens were coupled to carboxylated microspheres (beads) and used to measure antibodies in very small volumes of human saliva samples using a bead-based, solution-phase assay. Beads were coupled with antigens from Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I.1 and G II.4) and hepatitis A virus. To ensure that the antigens were sufficiently coupled to the beads, coupling was confirmed using species-specific, animal-derived primary capture antibodies, followed by incubation with biotinylated anti-species secondary detection antibodies and streptavidin-R-phycoerythrin reporter (SAPE). As a control to measure non-specific binding, one bead set was treated identically to the others except it was not coupled to any antigen. The antigen-coupled and control beads were then incubated with prospectively-collected human saliva samples, measured on a high throughput analyzer based on the principles of flow cytometry, and the presence of antibodies to each antigen was measured in Median Fluorescence Intensity units (MFI). This multiplex immunoassay has a number of advantages, including more data with less sample; reduced costs and labor; and the ability to customize the assay to many targets of interest. Results indicate that the salivary multiplex immunoassay may be capable of identifying previous exposures and infections, which can be especially useful in surveillance studies involving large human populations.

In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously.

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

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 ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...