A six‐color flow cytometric assay for the analysis of peripheral blood dendritic cells

Giannelli, Stefania; Taddeo, Adriano; Presicce, Pietro; Villa, Maria Luisa; Bella, Silvia Della

doi:10.1002/cyto.b.20434

Cited by 23 publications

(17 citation statements)

References 29 publications

(29 reference statements)

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“…The inclusion of both CD3 and CD4 antibodies is therefore important in staining of human PBMCs, to permit a better characterization of these CD8 + FOXP3 + cells. Although it has been shown that FMO controls allow to accurately determine positivity and set regions in samples containing multilabeled subpopulations (32,33,46–48), our results indicate that it can lead to overestimation of the percentage of FOXP3 + cells.…”

Section: Discussioncontrasting

confidence: 60%

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Association of two clones allows for optimal detection of human FOXP3

et al. 2010

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FOXP3 is a key transcription factor expressed by regulatory T cells (Treg cells). However, differences in staining and analysis protocols have led to conflicting results. Moreover, the transient upregulation of FOXP3 that follows activation in non-Treg cells renders the interpretation of FOXP3 data more difficult in humans than in mice. Human peripheral blood mononuclear cells (PBMCs), isolated CD25 2 or CD25 1 CD4 1 T cells were stained with three different anti-FOXP3 clones (PCH101, 206D, and 259D) alone or in combination, and using different permeabilization methods. REGULATORY T cells (Treg cells) play a major role in the homeostasis of the immune system (1-5). Treg cells are a subpopulation of CD4 1 T cells that represent $1-10% of circulating CD4 1 T cells (6). They were first characterized by the constitutive expression of the IL-2Ra chain (CD25) (1). More recently, it was shown that they usually express low levels of the IL-7Ra chain (CD127) (7,8). The transcription factor FOXP3 (Forkhead box P3) plays a crucial role in Treg differentiation, function, and biology in both mice and humans (9-13). However, because FOXP3 is an intracellular protein, surrogate surface markers must be used to purify Treg cells. Additional markers are frequently associated with human Treg function, including cytotoxic T lymphocyte associated antigen (CTLA-4) (14), L-selectin (CD62L) (15), aE-integrin (CD103) (16,17), and the glucocorticoid-induced tumor necrosis factor receptor (GITR) (18). However, none of these markers are selectively expressed by Treg cells, as they are also transiently upregulated in recently activated effector T (Teff) cells (18-21).The first commercially available monoclonal antibody (mAb) for studies of FOXP3 in human Treg cells (hFOXY) was quickly supplanted by the more specific mouse mAbs 206D, 236A, and 259D (22) and the rat anti-FOXP3 PCH101 clone (eBioscience) in flow-cytometry applications. PCH101 targets the N-terminal region of the 431 amino acid (aa) FOXP3 protein, while 206D, 236A, and 259D bind an epitope within the remaining N-terminus (aa 105-235), near the zinc finger region of

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Section: Discussioncontrasting

confidence: 60%

“…Compensation for 6 and 8 color stain sets was accomplished based on either antibody-capture beads (CompBeads, BD) (32,33) or PBMCs stained with the above described antibodies. Before experiments, PMT values were adjusted running unstained cells to exclude autofluorescence and to control the intensity of background.…”

Section: Methodsmentioning

confidence: 99%

Association of two clones allows for optimal detection of human FOXP3

et al. 2010

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“…In the growing trend for automation in the laboratory, the manual smear review represents one of the last steps to show improved productivity and achieve consistent quality of results. In the last two decades, flow cytometry has been proposed as the reference method for the detection of various cells in blood; for instance, immature granulocytes, lymphocytes, monocytes, and dendritic cells (8)(9)(10)(11)(12). However, all these studies were based on single cell type identification per antibody combination and were not suitable for a full differential count.…”

Section: Discussionmentioning

confidence: 99%

Refining the white blood cell differential: The first flow cytometry routine application

et al. 2010

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A Complete Blood Count performed by an automated hematology analyzer frequently needs a microscopic slide review. This step is time consuming and requires experienced personnel. Recently, several teams have proposed and validated convenient combinations of monoclonal antibodies for an extended white blood cell (WBC) differential by flow cytometry. The aim of this study was to evaluate the usefulness of this approach in the routine workflow of a hematology laboratory. We compared a workflow chain comprised of a robotic blood preparation system (for antibody labeling), a flow cytometer, and data management software to the standard manual review of a blood film and evaluated the diagnostic quality, the turnaround time, and the labor needed for the two different approaches. The study on 1,973 samples was organized, firstly, to determine analytic thresholds and these settings were then validated. The flow cytometric data management software automatically validated 52% of the samples without significant numbers of false negatives. Of the remaining specimens, an operator validated a further 33% of the samples and 15% needed a manual microscopic review. These results were obtained in a mean timeline similar to the traditional microscopic manual review. Our study demonstrates, for the first time, the efficiency of a flow cytometer integrated into a WBC differential workflow in a routine hematology laboratory. ' 2010 International Society for Advancement of Cytometry

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“…1 % of human CD45+ cells in the blood were mDC, whereas pDC represented 0.2 % of human CD45 + cells. Similarly, greater numbers of mDCs than pDCs have been noted in human peripheral blood [38, 39]. …”

Section: Generation Of Humanized Micementioning

confidence: 93%

Humanized mice: novel model for studying mechanisms of human immune-based therapies

2013

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The lack of relevant animal models is the major bottleneck for understanding human immunology and immunopathology. In the last few years, a novel model of humanized mouse has been successfully employed to investigate some of the most critical questions in human immunology. We have set up and tested in our laboratory the latest technology for generating mice with a human immune system by reconstituting newborn immunodeficient NOD/SCID-γc-/- mice with human fetal liver-derived hematopoietic stem cells. These humanized mice have been deemed most competent as human models in a thorough comparative study with other humanized mouse technologies. Lymphocytes in these mice are of human origin while other hematopoietic cells are chimeric, partly of mouse and partly of human origin. We demonstrate that human CD8 T lymphocytes in humanized mice are fully responsive to our novel cell-based secreted heat shock protein gp96HIV-Ig vaccine. We also show that the gp96HIV-Ig vaccine induces powerful mucosal immune responses in the rectum and the vagina, which are thought to be required for protection from HIV infection. We posit the hypothesis that vaccine approaches tested in humanized mouse models can generate data rapidly, economically and with great flexibility (genetic manipulations are possible), to be subsequently tested in larger nonhuman primate models and humans.

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A six‐color flow cytometric assay for the analysis of peripheral blood dendritic cells

Cited by 23 publications

References 29 publications

Association of two clones allows for optimal detection of human FOXP3

Association of two clones allows for optimal detection of human FOXP3

Refining the white blood cell differential: The first flow cytometry routine application

Humanized mice: novel model for studying mechanisms of human immune-based therapies

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