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Microvascular endothelial cells (MVECs) have been an important tool in many research fields, while their purification remained challenging, particularly from thin and fragile tissues, such as murine intestinal mucosae. Therefore, we established an immunomagnetic method for isolating rat intestinal mucosal MVECs using an automatic magnetic separation system from the primary cell culture, which was preliminarily purified by the differential adhesion combined with mechanically scraping method. The CD31

Neutrophil gelatinase-associated lipocalin (NGAL) is a promising biomarker for diagnosing acute kidney injury (AKI). Currently, there are few assays for determining NGAL and they are complex, time-consuming or expensive. We aimed to establish an efficient immunoassay to measure NGAL in human urine simply and rapidly. A novel immunoassay for NGAL determination was established by combining a dissociation-enhanced-free time-resolved fluoroimmunoassay (TRFIA) and immunomagnetic separation. Based on a “sandwich”-type immunoassay format, analytes in samples were captured by a pair of monoclonal antibodies (mAb) in which one mAb was coated in magnetic beads and the other mAb was labeled with europium(III) chelate microparticles (CM-EUs) as “fluorescent reporters”. NGAL concentrations were determined in a linear range (10–1500 ng mL−1) with a limit of detection of 0.32 ng mL−1. The reproducibility, recovery, and specificity of our TRFIA were acceptable. Our method was compared with that of a chemiluminescence immunoassay (CMIA) using 115 urine samples, and the results showed good correlation (R2 = 0.8677). We expect our novel method to be useful for the early diagnosis of AKI.

Sepsis is a systemic inflammatory response ensuing from presence and persistence of microorganisms in the bloodstream. The possibility to identify them at low concentrations may improve the problem of human health and therapeutic outcomes. So, sensitive and rapid diagnostic systems are essential to evaluate bacterial infections during the time, also reducing the cost. In this study, from random M13 phage display libraries, we selected phage clones that specifically bind surface of Staphyloccocus aureus, Pseudomonas aeruginosa and Escherichia coli. Then, commercial magnetic beads were functionalized with phage clones through covalent bond and used as capture and concentrating of pathogens from blood. We found that phage-magnetic beads complex represents a network which enables a cheap, high sensitive and specific detection of the bacteria involved in sepsis by micro-Raman spectroscopy. The enter process required 6 h and has the limit of detection of 10 Colony Forming Units on 7 ml of blood (CFU/7 ml).

Tumor endothelial cells (TECs), which constitute the lining of the tumor blood vessels, have various characteristics as tumor constituent cells. In this study, we describe a novel method for the isolation of highly pure, fresh TECs, which form a small population within the tumor. Tumors were first dissected from tumor-bearing mice and digested to a single cell suspension with Collagenase Type II; the single cells were then separated by density gradient centrifugation. TECs were enriched by CD31-positive selection using magnetic activated cell sorting and subsequently purified by fluorescence activated cell sorting. The high purity of the obtained cells was verified by flow cytometry. Upon cell culture, the isolated cells showed a polygonal shape and a cobblestone appearance, which are features of the endothelial cells. Furthermore, a functional assay revealed that the TECs suppressed the proliferation of CD8+ T cells in vitro. We believe that the isolation method described in this study will enable the further elucidation of the characteristics of TECs.

Objective In this study, a novel, simple, and rapid immunoassay for the determination of gastrin-17 (G-17) in human serum was established by combining immunomagnetic beads with time-resolved fluorescence immunoassay (TRFIA). Methods Immunomagnetic beads were coated with anti-G-17 M01 antibody, anti-G-17 M02 antibody was labeled with Eu3+ chelates. The concentration of G-17 in the serum was detected with the double-antibody sandwich method. Results The limit of background(LOB), limit of detection (LOD), and limit of quantification (LOQ) were 0.09, 0.104, and 0.39 pmol/L, respectively. The detection range of G-17-TRFIA was 0.39–100 pmol/L. The average intra- and inter-assay coefficients of variation (CV) were 5.95%–9.07% and 6.09%–8.14%, respectively. The recoveries for the serum samples ranged from 94.70% to 100.95%. The specificity of our G-17-TRFIA was acceptable. The correlation coefficient between G-17-TRFIA and commercial G-17-ELISA methods was R2 = 0.9092. Conclusions A novel G-17-TRFIA detection method was successfully established to provide a reference for the early diagnosis of patients with atrophic gastritis in clinical research.

The ability to quantify protein-ligand interactions in an accurate and high-throughput manner is important in diverse areas of biology and medicine. Multiplex bead binding assays (MBBAs) are powerful methods that allow for simultaneous analysis of many protein-ligand interactions. Although there are a number of well-established MBBA platforms, there are few platforms suitable for research and development that offer rapid experimentation at low costs and without the need for specialized reagents or instruments dedicated for MBBA. Here, we describe a MBBA method that uses low-cost reagents and standard cytometers. The key innovation is the use of the essentially irreversible biotin-streptavidin interaction. We prepared a biotin-conjugated fluorescent dye and used it to produce streptavidin-coated magnetic beads that are labeled at distinct levels of fluorescence. We show the utility of our method in characterization of phage-displayed antibodies against multiple antigens of SARS-CoV-2, which substantially improves the throughput and dramatically reduces antigen consumption compared with conventional phage ELISA methods. This approach will make MBBAs more broadly accessible.

Adoptive cell therapy with genetically modified regulatory T cells (Tregs) is under clinical investigation for the treatment of transplant rejection and various autoimmune conditions. A limitation of modelling this approach in mice is the lack of optimized protocols for expanding and transducing mouse Tregs. Here we describe a protocol for purifying, expanding and retrovirally transducing mouse Tregs with a vector encoding a chimeric antigen receptor as a model transgene. We found that isolation of Tregs from C57Bl/6J Foxp3EGFP mice solely based on eGFP expression resulted in sufficiently pure cells; co-sorting of CD25hi cells was not essential. Although expansion with rapamycin reduced Treg expansion, it promoted maximal in vitro suppressive activity. Retroviral transduction of Tregs following 2 days of stimulation with anti-CD3/CD28 beads achieved a transduction efficiency of ~40% and did not impair their suppressive capacity. When injected into a conventional T cell (Tconv)-transfer-induced colitis model, transduced Tregs inhibited colitis progression at ratios as low as 1 Treg to 100 Tconvs, and maintained Foxp3 and transgene expression throughout an 8-week period. This method facilitates the study of transduced Tregs in animal models and will enable the study of genetically engineered Treg therapy for a variety of inflammatory diseases.

This article aims to envisage future perspectives of the lymphocyte transformation test (LTT). We describe the select innovative techniques, which can be integrated at different stages of the LTT to potentially improve the sensitivity, specificity, or practicability of the LTT. We first focus upon the cell sorting techniques comprising immunomagnetic cell separation and flow cytometry, which can be implemented prior and after the LTT culturing step to concentrate and quantify specific immune cell types. Further, we elaborate upon three important omics techniques such as transcriptomics, proteomics, and metabolomics, which can be integrated downstream of the LTT to analyze molecular changes in specific immune cells following drug induced activation and proliferation. We also develop visions, how state of the art techniques used in other scientific fields, can be transferred and applied in the context of in-vitro detection of drug allergy.

Next generation sequencing (NGS) is widely applied in immunological research, but has yet to become common in antibody epitope mapping. A method utilizing a 12-mer random peptide library expressed in bacteria coupled with magnetic-based cell sorting and NGS correctly identified more than 75% of epitope residues on the antigens of two monoclonal antibodies (trastuzumab and bevacizumab). PepSurf, a web-based computational method designed for structural epitope mapping was utilized to compare peptides in libraries enriched for monoclonal antibody (mAb) binders to antigen surfaces (HER2 and VEGF-A). Compared to mimotopes recovered from Sanger sequencing of plated colonies from the same sorting protocol, motifs derived from sets of the NGS data improved epitope prediction as defined by sensitivity and precision, from 18% to 82% and 0.27 to 0.51 for trastuzumab and 47% to 76% and 0.19 to 0.27 for bevacizumab. Specificity was similar for Sanger and NGS, 99% and 97% for trastuzumab and 66% and 67% for bevacizumab. These results indicate that combining peptide library screening with NGS yields epitope motifs that can improve prediction of structural epitopes.

To establish a simple and widely accessible technique for rapidly selecting high producing Chinese hamster ovary (CHO) cells engineered to express a monoclonal antibody (mAb), we have exploited the transient display of recombinant protein on their cell surface. In combination with magnetic bead-based methods, we demonstrate the ability to select for cells of high productivity in the absence of any metabolic-based selection method. This technique is sufficient to obtain genetically stable engineered CHO cells via a single step of cell subcloning and yields sought-after stable, high IgG producing clonal cell lines. This technique may also be applied to other types of cells as well as polyclonal Ab cell pools.

Introduction Peripheral blood mononuclear cells (PBMCs) are frequently used for genomic analyses, but several factors can affect the yield and integrity of nucleic acids, including the methods of cell collection and isolation. The goal of this work was to analyze the utility of systematic isolation of different immune cell subsets by immunomagnetic separation and the RNA integrity after isolated cells from samples of HIV-infected patients. Methods PBMC from Healthy Controls (HC, n = 15), Elite Controllers (EC, n = 15), Viremic Controllers (VC, n = 15), Viremic Progressors (VP, n = 15) and HIV-infected patients on therapy (ART, n = 15) were isolated by Ficoll-Paque density gradient centrifugation. Subsets were separated with monoclonal antibodies (CD56, CD14, CD4, and CD8) conjugated to microbeads. We evaluated the yield and purity of each subset isolated from PBMCs under resting and activated conditions; LPS, anti-CD3/CD28 and anti-CD16 were used to activate monocytes, PBMC, T cells and NK cells, respectively. The quality of extracted RNA was tested by 2100 Bioanalyzer. Results In resting conditions, the average yield of CD14 + (monocytes) was decreased (p = 0.021) in HIV + patients compared with healthy controls. CD56 + (Natural Killer-NKs; p = 0.03) and CD8 + (Cytotoxic T lymphocytes-CTL p = 0.001) cells were increased in HIV + patients after 72 h of activation. The purity assay detected significant differences in CD14 + (p ≤ 0.001) and CD8 + (p = 0.034) subpopulations when comparing PBMC isolated either from healthy controls or HIV + patients. The number of activated cells in HIV + presented differences in CD8 subset (p = 0.003). Finally, similar quantities of high quality RNA were extracted from immune cells subsets obtained by our method. Specifically, we show that Bioanalyzer electrophenograms reveal optimal RIN values in HIV positive and negative patients in resting condition (EC:8;HC:6.5;VC:8.80;VP:8;HAART:7.5) and activated condition (EC:9;HC:6.7;VC:8.2;VP:7.2;HAART:8.6). Conclusion This method allowed us to obtain a sufficient quantity of different isolated immune cell subsets from HIV–infected individuals at different disease stages. Moreover, the assessed qualities of nucleic acids allow us to perform subsequent molecular studies, such as microRNA profiling.

Dendritic cells (DCs) are increasingly being used for multiple applications and are useful tools for many immunotherapeutic strategies. The understanding of the possible impact of the DCs-generation methods on the biological capacities of these cells is therefore essential. Although the immunomagnetic separation is regarded as a fast and accurate method yielding cells with the high purity and efficiency, still little is known about its impact on the properties of the generated DCs. The aim of this study was to compare the morphology of the monocyte derived dendritic cells (MoDCs), generated from monocytes selected with anti-CD14 mAbs (positive separation) and treated with anti-CD3, -CD7, -CD16, -CD19, -CD56, -CD123, glycophorin A (negative separation), using laser scanning microscopy. We found that the type of the immunomagnetic separation method used strongly influences the shape and cell dimension of the MoDCs. We observed that the height of both immature and LPS-matured DCs generated from monocytes isolated by negative separation was significantly higher compared to the cells obtained by positive separation.

Multiplex bead-based flow cytometry is an attractive way for simultaneous, rapid and cost-effective analysis of multiple analytes in a single sample. Previously, we developed various bead-based assays using non-magnetic beads coated with Staphylococcus aureus and Streptococcus pneumoniae antigens for the detection of antibodies. Here, we compared the performance of the assay using non-magnetic beads with one based on the newly developed magnetic beads. We optimized the magnetic beads' coupling procedure and antibody detection assays for S. aureus and S. pneumoniae antigens and we measured IgG in human pooled serum against a series of S. aureus and S. pneumoniae-derived antigens in a singleplex and in a multiplex assay, respectively. For the multiplex assay, the comparison between magnetic and non-magnetic beads showed: i) in the majority of the cases (13 of the 17 tested S. pneumoniae antigens) significantly higher Median Fluorescence Intensity (ME) values, ii) lower detection limits, iii) lower coefficient of variation (CV: 12% vs. 7% for non-magnetic vs. magnetic beads), so lower inter assay variation and hence higher reproducibility. Magnetic bead coupling is cost effective, as we used 25% of the normal amount of antigen and only 50% of the beads in comparison to the non-magnetic beads. This optimized magnetic-based assay, which combines ease of use with an improved assay performance, allows detection of antibodies with a low titer that are potentially missed with the non-magnetic-based assay. (C) 2016 Elsevier B.V. All rights reserved.

Microglia and astrocytes, the two innate cells in CNS, are thought to protect and remodel of synapses for proper maintenance and plasticity of neuronal circuits. The two types of cells are the major responders by producing and releasing inflammatory mediators. Isolation of microglia and astrocytes from CNS tissue provides a powerful tool to study basic cell biology and examine the effects of in vivo treatments on microglia and astrocytes immunophenotype and function. The widely used approach of enrichment microglia and astrocytes from CNS was MACS (Magnetic activated cell sorting) and FACS (Fluorescence activated cell sorting). Here we described an optimized protocol of enzymatic dissociation generating single cell suspensions from brain tissue. Then the ability of the two methods to isolate microglia and astrocytes from brain dissociated cells was compared. Both MACS and FACS processing could obtain microglia and astrocytes with high viability (>85%). Microglia sorted by MACS comprises a slight myeloid cells contamination but with a little bit higher efficiency than that sorted by FACS. MACS processing was faster than FACS for either single or multiple samples. ACSA2 can be used to isolate astrocytes from both postnatal and adult brain, and is more suitable for purify astrocytes from newborn. FACS could get purer microglia which is helpful for deep sequencing and other related research. ACSA2 is a good marker of astrocytes.

Cell-based assays to monitor antigen-specific T-cell responses are characterized by their high complexity and should be conducted under controlled conditions to lower multiple possible sources of assay variation. However, the lack of standard reagents makes it difficult to directly compare results generated in one lab over time and across institutions. Therefore TCR-engineered reference samples (TERS) that contain a defined number of antigen-specific T cells and continuously deliver stable results are urgently needed. We successfully established a simple and robust TERS technology that constitutes a useful tool to overcome this issue for commonly used T-cell immuno-assays. To enable users to generate large-scale TERS, on-site using the most commonly used electroporation (EP) devices, an RNA-based kit approach, providing stable TCR mRNA and an optimized manufacturing protocol were established. In preparation for the release of this immuno-control kit, we established optimal EP conditions on six devices and initiated an extended RNA stability study. Furthermore, we coordinated on-site production of TERS with 4 participants. Finally, a proficiency panel was organized to test the unsupervised production of TERS at different laboratories using the kit approach. The results obtained show the feasibility and robustness of the kit approach for versatile in-house production of cellular control samples.

Antibodies labeled with small molecules such as fluorophore, biotin or drugs play an important role in various areas of biological research, drug discovery and diagnostics. However, the majority of current methods for labeling antibodies is solution-based and has several limitations including the need for purified antibodies at high concentrations and multiple buffer exchange steps. In this study, a method (on-bead conjugation) is described that addresses these limitations by combining antibody purification and conjugation in a single workflow. This method uses high capacity-magnetic Protein A or Protein G beads to capture antibodies directly from cell media followed by conjugation with small molecules and elution of conjugated antibodies from the beads. High-capacity magnetic antibody capture beads are key to this method and were developed by combining porous and hydrophilic cellulose beads with oriented immobilization of Protein A and Protein G using HaloTag technology. With a variety of fluorophores it is shown that the on-bead conjugation method is compatible with both thiol- and amine-based chemistry. This method enables simple and rapid processing of multiple samples in parallel with high-efficiency antibody recovery. It is further shown that recovered antibodies are functional and compatible with downstream applications.

Clinically relevant and reliable reports derived from in vitro research are dependent on the choice of cell isolation protocols adopted between different laboratories. Peripheral blood eosinophils are conventionally isolated using density-gradient centrifugation followed by immunomagnetic selection (positive/negative) while neutrophils follow a more simplified dextran-sedimentation methodology. With the increasing sophistication of molecular techniques, methods are now available that promise protocols with reduced user-manipulations, improved efficiency, and better yield without compromising the purity of enriched cell populations. These recent techniques utilize immunomagnetic particles with multiple specificities against differential cell surface markers to negatively select non-target cells from whole blood, greatly reducing the cost/time taken to isolate granulocytes. Herein, we compare the yield efficiencies, purity and baseline activation states of eosinophils/neutrophils isolated using one of these newer protocols that use immunomagnetic beads (MACSxpress isolation) vs. the standard isolation procedures. The study shows that the MACSxpress method consistently allowed higher yields per mL of peripheral blood compared to conventional methods (P

Magnetic activated cell sorting (MACS) is a commonly used cell separation technique. Miltenyi Biotec has developed an efficient MACS protocol for isolation of CD8α(+) T cells from adult mice spleen. However, due to a low percentage of CD8α(+) T cells in spleen of postnatal mice, MACS isolation of CD8α(+) T cells results in a low purity. Our study illustrates how this problem can be solved by performing a CD45R(+) B cell depletion prior to a positive selection of CD8α(+) T cells. This protocol can be used when low frequencies of CD8α(+) T cells are present in a tissue also populated with CD45R(+) B cells, and a high purity is required for downstream applications.

Functional studies of cellular immunity in patients with leukemia often require separation of leukemic cells from other peripheral blood mononuclear cells (PBMCs). This can pose a challenge when the number of leukemic cells is very high, such as in untreated patients with chronic lymphocytic leukemia (CLL). We found that when leukemia cell frequency was very high, anti-CD19 coated immunomagnetic beads did not thoroughly deplete B cells when used according to manufacturer's instructions. In this study, we depleted leukemic B cells using a modified protocol comprising serial rounds of depletion using immunomagnetic beads at reduced bead to cell ratios. This resulted in more effective B cell depletion with the use of fewer immunomagnetic beads, and without affecting viability or yield of non-B cells. CD19 − PBMC subsets were retained, and serial depletion rounds did not activate T cells and monocytes. The positively isolated CLL cells were of high purity and were available for downstream analysis. This is a convenient and cost-effective method to enable in vitro analysis of immunocompetent cells from patients with leukemia.

Ex-vivo differentiation of regulatory T cells (Tregs) from naive CD4+ T-cells has been widely used in immunological research. Isolation of a highly pure naive T cell population is the key factor that determines the efficiency of subsequent Treg differentiation. Currently, this step relies mostly on FACS sorting, which is often costly, time consuming, and inconvenient. Alternatively, magnetic separation of T-cells can be performed; yet, available protocols fail to reach sort level purity and consequently result in low Treg differentiation efficiency. Here, we present the results of a comprehensive side-by-side comparison of various magnetic separation strategies and FACS sorting in multiple levels. Additionally, we propose a novel optimized custom made magnetic separation protocol, which not only yields sort level purity and Treg differentiation but also lowers the reagent costs up to 75% compared to the commercially available purification kits. The highly pure naive CD4+ T-cell population obtained by this versatile method can also be used for differentiation of other T-cell subsets; therefore this protocol may have broad applications in T-cell research.

Here we describe a new analytical method for the detection of two influenza A viruses by nitrocellulose membrane and magnetic sensors that employ a special frequency mixing technique. The combination of the nitrocellulose membrane and magnetic bead detection permits a rapid assay procedure and excludes two steps (the development of color and the stop reaction) required for usual immunochemical detection methods such as ELISA. Quantitative virus detection was performed using magnetic beads conjugated with secondary antibody. The results were compared with conventional assay methods and with a dot-blot assay with fluorescence compound (FITC). Under optimum conditions, our new assay procedure is capable of detecting picograms of virus per well. This new method combining the nitrocellulose membrane and magnetic bead detection reduces analytical time and allows stable and repeatable analyses of samples in point-of-care applications.

Adipose tissue has long been considered to be a simple tissue that contains adipocytes. Because of this, the isolation and characterization of microvascular endothelical cells, which are also present in adipose tissue, have been neglected, even though they are components of a capillary network that surrounds each individual adipocyte. Here we report on a protocol for producing highly purified murine microvascular endothelial cells (MECs) from diverse sites of murine adipose tissues including inguinal and epididymal adipose tissues. The method is based on a combination of negative and positive immunomagnetic selection. The protocol involves the preparation of a single cell suspension (digestion, filtration and density gradient centrifugation), immunomagnetic enrichment of the CD45− cell population and the purification of the MECs by a combination of common specific markers CD31, CD102 and isolectin B4. The isolated MECs can be successively cultured for 10 to 12 passages without any detectable changes in morphology and phenotype. Therefore, the method described herein represents a protocol for the isolation and long-term maintenance of highly pure mouse MECs in high yields from adipose tissues.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) utilizes antibodies to enrich peptides from complex matrices for quantitation by stable isotope dilution mass spectrometry. SISCAPA improves sensitivity and limits the sample handling required for plasma-based analysis. Thus far, SISCAPA assays have been performed using polyclonal antibodies, yet monoclonal antibodies are an attractive alternative since they provide exquisite specificity, a renewable resource, and the potential for isolation of clones with very high affinities (10−9 M or better). The selection of a good monoclonal antibody out of hundreds-to-thousands of clones presents a challenge, since the screening assay should ideally be in the format of the final SISCAPA assay, but performing the assays manually is labor- and time-intensive. In this manuscript, we demonstrate that monoclonal antibodies can be used in SISCAPA assays, and we describe an automated high-throughput SISCAPA method that makes screening of large numbers of hybridomas feasible while conserving time and resources.

Autoantibodies are central to the diagnosis and assessment of systemic lupus erythematosus (SLE). A recent technique for the measurement of autoantibodies utilizes addressable laser bead immunoassay technology (BioPlex 2200) which permits the simultaneous detection of multiple autoantibodies and improved efficiency due to the shorter time to perform the assay and low volume of test samples and reagents. In the current study we have compared this technique to more traditional measures of autoantibody detection. The clinical and laboratory data and stored serum samples from the enrollment visit into a long-term lupus registry at a single academic medical center were used. Sera were examined for a panel of autoantibodies using the BioPlex ANA screen. The results were compared to the historical data on autoantibody profiles using indirect immunofluorescence (IIF) and ELISA. The association with global and organ specific SLE disease activity (nephritis) was also examined. The study consisted of 192 patients who were predominantly female (87%) and Caucasian (91%) with mean disease duration of 8.8 years. The frequency of ANA and anti-dsDNA by IIF and ELISA was 81.3% and 46.6% respectively and was higher than that found with BioPlex (75.5% and 31.8%). The latter detected a higher proportion of patients with autoantibodies to Sm (7.5% vs 16.7%), RNP (21.8% vs 24.0%), Ro (37.4% vs 41.7) and La (13.9% vs 23.4%). Overall agreement between assays varied between 71.4% and 92.5%. Additional autoantibodies identified by BioPlex were anti-chromatin antibodies which were similar in frequency to anti-dsDNA antibodies (33.9% and 31.8% respectively). There was a low frequency of anti-ribosomal P (6.8%), anti-Scl-70 (5.2%), anti-centromere B (3.7%) and anti-Jo-1 (0.5%). Several autoantibodies revealed significant associations with SLEDAI scores but in a multivariate analysis the only autoantibodies that approached statistical significance were anti-Sm (p=0.094) measured by ELISA and anti-dsDNA (p=0.082) measured by BioPlex. There was no association between any of the autoantibodies regardless of the method of detection and cumulative organ damage scores. Fifty-three patients (27.6%) had lupus nephritis of which 17 (32%) had active nephritis at the time of autoantibody determination. There was no significant association between a positive ANA (IIF) and any autoantibodies detected by ELISA with either the cumulative occurrence of lupus nephritis or active nephritis. In contrast, there was an association between BioPlex detected anti-dsDNA with the cumulative occurrence of nephritis (p=0.074) which reached statistical significance with active nephritis at the time of antibody testing (p=0.012). This was confirmed by multivariate analysis (p=0.047). These results suggest reasonable agreement between the detection of lupus autoantibodies by ELISA and BioPlex. The latter demonstrated a better correlation with lupus nephritis.

Interest in gamma9delta2 T cells has increased greatly in the past decade. While several protocols allowed the amplification of a large proportion of these cells in vitro, the purity of the final preparation is usually heterogeneous between different donors. Functional studies of this population are often controversial due to the presence of other populations such as NK cells which share a wide range of characteristics. Here, the gamma9delta2 T cells labelled-fraction is purified and mixed with the irradiated unlabelled fraction followed by a single stimulation with phosphoantigen, in turn followed by a classical step of amplification in the presence of interleukin 2. In this study, we describe a straightforward protocol to amplify pure populations of gamma9delta2 T cells which could be useful in fundamental research or in the development of a new generation of gammadelta cell therapy protocol.

To date, studies on T cells in acute myeloid leukemia (AML) have been limited to flow cytometric analysis of whole peripheral blood mononuclear cell (PBMC) specimens or functional work looking at the impact of AML myeloblasts on normal or remission T cells. This lack of information on T cells at the time of presentation with disease is due in part to the difficulty in isolating sufficiently pure T cells from these specimens for further study. Negative immunomagnetic selection has been the method of choice for isolating immune cells for functional studies due to concerns that binding antibodies to the cell surface may induce cellular activation, block ligand-receptor interactions or result in immune clearance. In order specifically to study T cells in presentation AML specimens, we set out to develop a method of isolating highly pure CD4 and CD8 T cells by negative selection from the peripheral blood (PB) of newly diagnosed AML patients. This technique, unlike T cell selection from PB from normal individuals or from patients with chronic lymphocytic leukaemia, was extremely problematic due to properties of the leukaemic myeloblasts. A successful method was eventually optimized requiring the use of a custom antibody cocktail consisting of CD33, CD34, CD123, CD11c and CD36, to deplete myeloblasts.

Human regulatory T cells (Treg) have been variously defined as CD4(+)CD25(+), CD4(+)CD25(high) or CD4(+)CD25(high)FOXP3(+) cells which are responsible for maintaining peripheral tolerance. Their isolation from human peripheral blood or tissues depends on the expression level of CD25(IL-2Ralpha) - a surface marker which is also expressed on activated effector helper T cells. CD39, a cell surface associated ectonucleotidase, can be used to purify Treg with strong suppressor functions. The CD4(+)CD39(+) T cells catalyze cleavage of adenosine triphosphate (ATP) to adenosine monophosphate (AMP), which is then further cleaved to adenosine. CD4(+)CD39(+) T cells largely overlap with CD4(+)CD25(high)FOXP3(+) but not CD4(+)CD25(+) T cell subset, and mediate equally potent immune suppression. Thus, CD39 surface marker can be successfully used for routine isolation of functionally-active human Treg from the peripheral blood of healthy donors or patients with cancer for studies of their role in health and disease.

Current methods for the detection and isolation of antigen-specific CD4 + and CD8 + T cells require the availability of peptide/MHC multimers or are restricted to cells that produce cytokines after antigen contact. Here we show that de novo cell surface expression of the TNF-receptor family member CD137 (4-1BB) identifies recently activated, but not resting, human CD4 + and CD8 + memory T cells. Maximum CD137 expression level is uniformly observed in both T-cell subsets at 24h after stimulation with antigen. In experiments with CMV and EBV-reactive T cells, we confirmed the specificity of CD137 expression by co-staining with peptide/HLA tetramers. Substantial proportions of CD137 + T cells did not produce IFN-γ, suggesting that CD137 detects a broader repertoire of antigen-specific T cells. Activated CD137 + T cells could be easily purified by MACS and expanded in vitro thereafter. This CD137-based enrichment method was capable of isolating 2-fold higher numbers of anti-viral CD4 + and CD8 + T cells compared to the IFN-γ secretion assay. In conclusion, antigen-triggered CD137 expression allows the rapid detection and sorting of virus-reactive CD4 + and CD8 + T cells. The CD137 assay is most attractive for the simultaneous targeting of anti-viral T helper and effector cells in monitoring studies and adoptive immunotherapy trials.

E. coli O157:H7 is an enterohemorrhagic bacteria that cause deadly water-borne infections implicated in outbreaks of a wide spectrum of human gastrointestinal diseases. It is therefore important to have a rapid convenient, simple and sensitive range of detection of E. coli O157:H7. A new E. coli O157 MAb designated P124 was developed for ultrasensitive detection of E. coli O157 in water, apple juice and beef for routine use. A prototype filter dot assay was designed with anti-E. coli O157 MAb bound to 0.2 microm nitrocellulose filter disk as the capture antibody. A 100 ml water sample spiked with 1-50 CFU of E. coli O157 either in the presence or absence of other non-specific bacteria were filtered for capture of the pathogen on the antibody coated nitrocellulose disk. The detection of the pathogen was successfully accomplished by the same antibody both as a capture and detecting antibody as a homosandwich. In a non-enriched format, detection of E. coli was possible with a sensitivity of 2500 CFU/100 ml. Ultrasensitive detection of ~1 CFU/100 ml sample could be achieved by a prior pathogen enrichment step before the addition of the labeled antibody. The design of this diagnostic test is based on the common architecture of all bacteria, viruses and spores, namely the manifestation of repeat lipopolysaccharide epitopes on the surface. We have developed an easy-to-use two dot visual filter assay for translation into current water testing in public health laboratories to detect E. coli O157:H7. In a 5 h assay approximately 1 CFU and approximately 5 CFU of E. coli O157 could be detected in 100 ml of water or juice and lake samples respectively. This simple homosandwich enrichment strategy can also be used to detect low levels of other water-borne pathogens.

T helper (Th) 17 cells are a distinct lineage of CD4+ T cells mediating tissue inflammation through the secretion of IL-17. In addition, it has been shown that the expression of the transcriptional factor RORgammat is responsible for the induction and maintenance of this cell line. Th17 cells are believed to be involved in a variety of autoimmune disorders, but may also play an important role in host defense. Here we describe a novel technique to reproducibly isolate viable Th17 cells based on their IL-17 secreting ability. We confirmed Th17 cell enrichment by quantitative PCR analysis and demonstrate that positively selected cells using this technique express significantly increased mRNA levels of RORgammat, IL-23 receptor and CCR4 when compared to negatively selected cells. Furthermore, we show that purified Th17 cells can be maintained in long-term culture and expand in vitro. In conclusion, this technique will allow for the first time the direct, ex vivo analysis of phenotypic and functional properties of Th17 cells.

Interest in plasma cells has increased greatly in the past decade. While several studies have examined the longevity and transcriptional control of antibody secreting cells in vivo, few studies have examined freshly isolated plasma cells ex vivo. Studies of primary plasma cells have been limited primarily due to the difficulty of isolating the large numbers of plasma cells necessary for experiments. In this protocol, plasma cells are purified from murine bone marrow in approximately 5 hours, following a two-step isolation method utilizing column enrichment and FACS-sorting based on CD138 (Syndecan-1) surface expression.

Detection of cytotoxic activity by pathogen-specific T cells of unknown antigenic specificity is difficult due to the limitations of using infected cells, instead of peptide-pulsed cells, as targets. We report here that the recently described CD107 mobilization assay readily allowed for the ex vivo detection of cytotoxic T lymphocytes (CTL) with a novel MHC-Ib restriction that specifically recognized Listeria monocytogenes-infected macrophages. The CD107 mobilization assay is likely to be a useful tool for detection of CD8(+) T cells that recognize a wide variety of intracellular pathogens.

Neutrophils are phagocytic leukocytes that represent one of the first lines of defense during infection and injury. Neutrophils emigrate into tissues during inflammation and are phenotypically different compared to cells in the circulation. To further understand the biology of tissue-recruited neutrophils, we have developed a reliable method to isolate these cells from inflamed liver. Acute liver inflammation was induced in mice by systemic treatment with adenovirus vectors. Two hours following adenovirus treatment, livers were enzymatically digested and leukocytes isolated by Percoll density gradient centrifugation. Neutrophils were then purified by negative immunomagnetic separation. Neutrophils isolated in this manner were 95% pure as determined by flow cytometry and more than 97% viable by propidium iodide staining. In order to carry out molecular studies, we extracted high quality genomic DNA and RNA from isolated neutrophils. PCR was used to successfully amplify sample genes from isolated neutrophil DNA. Isolated neutrophil RNA was used in a ribonuclease protection assay to evaluate chemokine gene expression. Neutrophils were shown to express multiple chemokine mRNA transcripts including MIP-1 beta, MIP-2 and IP-10. This work describes a novel method to isolate highly pure, viable neutrophils from pathologically inflamed tissue for subsequent detailed cellular and molecular analysis.

Identification of key factors mediating the differentiation of naive CD4 + T helper cells into Th1 and Th2 subsets is important for understanding the molecular mechanisms of the development of autoimmune diseases as well as asthma and allergy. Functional importance of a given gene in the initiation of human T helper cell differentiation has been hard to study due to the difficulty in transfecting primary resting human T lymphocytes. In this study we have successfully transfected human primary CD4 + T helper cells using Amaxa's Nucleofection technology. To overcome the background caused by untransfected cells, we have developed a system for enriching nucleofected unstimulated human primary T helper cells that express the gene of interest. This is achieved by introducing a plasmid construct containing a bicistronic unit coding for a truncated mouse MHC class l H-2K k cell surface marker followed by selection of H-2K k positive cells using antibody coated beads. We demonstrate that the nucleofected and enriched H-2K k positive T helper cells differentiate into Th1 and Th2 cells as well as the non-transfected control cells. We also show that by using this novel method, introduction of an shRNA targeting Stat6, a key molecule driving the Th2 cell development, results in impaired Th2 cell differentiation, as expected. The method described here, enables fast and feasible preparation of highly pure transfected primary CD4 + T cell cultures ideal for studying the influence of overexpression or knockdown of a given gene on T helper cell differentiation and other primary human T cell functions.

In compliance with federal regulations, blood banks routinely use leukocyte depletion filters to eliminate contaminating leukocytes from blood products such as red blood cell and platelet concentrates. We developed and optimized conditions to elute leukocytes adsorbed to these filters; resulting in leukocyte suspensions which we termed Filter Buffy Coats (FBCs). These Filter Buffy Coats can replace standard buffy coats for various research applications. After optimizing both the filter elution medium as well as elution protocols, we compared commonly used leukocyte depletion filters from four different manufacturers. Relative fractions as well as total recoveries of leukocyte subsets, such as lymphocytes, monocytes and granulocytes, found in Filter Buffy Coats were identified and compared among the filters as well as to standard buffy coats and whole blood. Flow cytometric analysis of Filter Buffy Coats confirmed the presence of T- and B-lymphocytes, NK cells and monocytes. Furthermore, a significant quantity of CD34(+) hematopoietic stem or progenitor cells (HSC/HPC) was detected in Filter Buffy Coats prepared from different filters, thus making FBCs a valuable source for research on HSC/HPC. Colony assays revealed that most of these CD34(+) cells are functional. Using immunomagnetic cell sorting (MACS), we isolated a variety of leukocyte populations from FBC mononuclear cells (Filter-PBMCs) including T lymphocytes (CD4(+), CD8(+), CD3(+)), B lymphocytes (CD19(+)), NK cells (CD56(+)), HSC/HPC (CD34(+), CD133(+)) or dendritic cells (BDCA-4(+)). Functional properties of Filter-PBMCs, as well as of some of these isolated leukocyte populations, were confirmed using standard assays. In summary, Filter Buffy Coats are a valuable and convenient source of different peripheral leukocyte populations and can replace standard buffy coat preparations for research applications.

Both monoclonal (e.g. Orthoclone (OKT3), rituximab) and polyclonal (e.g. ATGAM, Thymoglobulin (Thymo)) anti-lymphocyte Abs (ALAs) are used extensively in organ transplantation for immunosuppression induction, desensitization, and treatment of acute rejection. ALAs often interfere with post transplant immunologic monitoring. We describe a method that uses magnetic beads to selectively remove ALAs from patient serum. Rabbit anti-mouse Fc-specific (180 mug), or rabbit anti-mouse Fab-specific (180 microg), or rabbit anti-horse heavy and light chain-specific and rabbit anti-horse F(ab')2 (200 microg) (Jackson Immunoresearch) was adsorbed to 6.7 x 10(8) Dynabeads M-280 conjugated with sheep anti-rabbit IgG (Dynal Biotech). Fifty microliters of normal human serum (NHS) with 2 microg/ml of OKT3 or 100 microg/ml ATGAM, Thymo, or rituximab were incubated with conjugated beads for several incubations. NHS containing ALAs before and after treatment by the protocol were incubated with human lymphocytes and labeled with FITC-antibody to immunoglobulin of the species used to produce the particular ALA. Residual ALA was determined using flow cytometry. Average median channel for serum with or without ALA was 11.1 and 0.120, respectively for OKT3; 64.4 and 0.344 for ATGAM; 108.5 and 0.200 for Thymo; and 1022.5 and 11.4 for rituximab. Treatment lowered the median channel for serum with OKT3 to 0.103, 0.309 for ATGAM, 0.199 for Thymo, and 12.1 for rituximab. ALAs can be effectively removed from serum by the use of magnetic beads conjugated with Ab specific for ALA thereby permitting immunologic monitoring without interference.