An 11-color flow cytometric assay for identifying, phenotyping, and assessing endocytic ability of peripheral blood dendritic cell subsets in a single platform

Wang, Jyh-Chiang E.; Kobie, James J.; Zhang, Li; Cochran, Matthew; Mosmann, Tim R.; Ritchlin, Christopher T.; Quataert, Sally A.

doi:10.1016/j.jim.2008.11.002

Cited by 26 publications

(30 citation statements)

References 29 publications

(40 reference statements)

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“…In order to further assess the function of mDCs, we measured antigen uptake by using the FITC-dextran uptake assay (32). Representative flow cytometry plots are shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…After resting, 2 ϫ 10 6 to 4 ϫ 10 6 PBMCs were left unstimulated for 24 h and for simplicity are referred to here as ex vivo PBMCs. To evaluate endocytosis, unstimulated cells were then incubated with FITC-dextran as previously described (32). Briefly, PBMCs were washed, resuspended at 20 ϫ 10 6 cells/ml in phosphate-buffered saline (PBS), 1% FBS, and distributed in two 96-well U-bottom plates before adding 1 mg/ml FITC-dextran (molecular weight, 40,000; Life Technologies).…”

Section: Methodsmentioning

confidence: 99%

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Sustained Hyperresponsiveness of Dendritic Cells Is Associated with Spontaneous Resolution of Acute Hepatitis C

Pelletier

Bédard

Said

et al. 2013

J Virol

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Some studies have reported that dendritic cells (DCs) may be dysfunctional in a subset of patients with chronic hepatitis C virus (HCV) infection. However, the function of DCs during acute HCV infection and their role in determining infectious outcome remain elusive. Here, we examined the phenotype and function of myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) during acute HCV infection. Three groups of injection drug users (IDUs) at high risk of HCV infection were studied: an uninfected group, a group with acute HCV infection with spontaneous resolution, and a group with acute infection with chronic evolution. We examined the frequency, maturation status, and cytokine production capacity of DCs in response to the Toll-like receptor 4 (TLR4) and TLR7/8 ligands lipopolysaccharide (LPS) and single-stranded RNA (ssRNA), respectively. Several observations could distinguish HCV-negative IDUs and acute HCV resolvers from patients with acute infection with chronic evolution. First, we observed a decrease in the frequency of mature CD86؉ , programmed death-1 receptor ligand-positive (PDL1 ؉ ), and PDL2 ؉ pDCs. This phenotype was associated with the increased sensitivity of pDCs from resolvers and HCV-negative IDUs versus the group with acute infection with chronic evolution to ssRNA stimulation in vitro. Second, LPS-stimulated mDCs from resolvers and HCV-negative IDUs produced higher levels of cytokines than mDCs from the group with acute infection with chronic evolution. Third, mDCs from all patients with acute HCV infection, irrespective of their outcomes, produced higher levels of cytokines during the early acute phase in response to ssRNA than mDCs from healthy controls. However, this hyperresponsiveness was sustained only in spontaneous resolvers. Altogether, our results suggest that the immature pDC phenotype and sustained pDC and mDC hyperresponsiveness are associated with spontaneous resolution of acute HCV infection.A minor fraction of individuals infected with the hepatitis C virus (HCV) eliminate the virus spontaneously, while the majority develop persistent infection, which may lead to chronic liver diseases (1). Spontaneous resolution of acute HCV infection is associated with the development of a robust and sustained HCV-specific CD4 and CD8 T cell response (2). In contrast, such responses are weak, inefficient, or transient in individuals who develop chronic infection. Several mechanisms underlying this immune failure have been previously described. First, escape mutations in epitopes targeted by virus-specific CD8 ϩ T cells can occur early during acute HCV infection, and this correlates with virus persistence (3-6). Second, failure of HCV-specific CD4 helper T cells to proliferate and produce cytokines was associated with viral recurrence and persistence. This loss of CD4 T cell help may compromise the function of virus-specific CD8 T cells and contribute to the development of suboptimal or exhausted CD8 ϩ T cells (7-13). Dendritic cells (DCs) are the most potent antigen-presenting cells in the body, and ...

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“…In order to further assess the function of mDCs, we measured antigen uptake by using the FITC-dextran uptake assay (32). Representative flow cytometry plots are shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Sustained Hyperresponsiveness of Dendritic Cells Is Associated with Spontaneous Resolution of Acute Hepatitis C

Pelletier

Bédard

Said

et al. 2013

J Virol

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“…Cells were centrifuged and re‐suspended in RPMI 1640, supplemented with 8% FBS. PBMC were labeled with a 15‐color T cell phenotyping panel of antibodies (Supporting Information Table 2) using a micromethod 12, 13. Cell data were acquired using an LSR II cytometer (BD Immunocytometry Systems).…”

Section: Methodsmentioning

confidence: 99%

Competitive SWIFT cluster templates enhance detection of aging changes

Rebhahn

Roumanes

et al. 2015

Cytometry Pt A

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Clustering‐based algorithms for automated analysis of flow cytometry datasets have achieved more efficient and objective analysis than manual processing. Clustering organizes flow cytometry data into subpopulations with substantially homogenous characteristics but does not directly address the important problem of identifying the salient differences in subpopulations between subjects and groups. Here, we address this problem by augmenting SWIFT—a mixture model based clustering algorithm reported previously. First, we show that SWIFT clustering using a “template” mixture model, in which all subpopulations are represented, identifies small differences in cell numbers per subpopulation between samples. Second, we demonstrate that resolution of inter‐sample differences is increased by “competition” wherein a joint model is formed by combining the mixture model templates obtained from different groups. In the joint model, clusters from individual groups compete for the assignment of cells, sharpening differences between samples, particularly differences representing subpopulation shifts that are masked under clustering with a single template model. The benefit of competition was demonstrated first with a semisynthetic dataset obtained by deliberately shifting a known subpopulation within an actual flow cytometry sample. Single templates correctly identified changes in the number of cells in the subpopulation, but only the competition method detected small changes in median fluorescence. In further validation studies, competition identified a larger number of significantly altered subpopulations between young and elderly subjects. This enrichment was specific, because competition between templates from consensus male and female samples did not improve the detection of age‐related differences. Several changes between the young and elderly identified by SWIFT template competition were consistent with known alterations in the elderly, and additional altered subpopulations were also identified. Alternative algorithms detected far fewer significantly altered clusters. Thus SWIFT template competition is a powerful approach to sharpen comparisons between selected groups in flow cytometry datasets. © 2015 The Authors. Published Wiley Periodicals Inc.

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“…Often, these exclusion markers are all grouped in one single channel, referred to as a dump channel, and, thus, it is impossible to also identify lymphocyte and monocyte populations (35,36). With our 12-color panel, we used the exclusion markers in different fluorescent channels, and thus were also able to assess the major lymphocytic populations (T, B, NK, and NKT cells) and monocyte subsets.…”

Section: Original Articlementioning

confidence: 99%

Evaluation of a 12‐color flow cytometry panel to study lymphocyte, monocyte, and dendritic cell subsets in humans

et al. 2010

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Monitoring changes in human immune cell populations such as lymphocytes, monocytes, and dendritic cells (DCs) during infectious diseases like human immunodeficiency virus (HIV) is crucial. However, difficulties to identify rare or heterogeneous cell populations can be limiting. For example, to accurately measure DC subsets, eight flow cytometry parameters are ideal. The aim of this work was to analyze the phenotype of human lymphocyte, monocyte, and DC subsets using a single 12-color flow cytometry panel. After erythrocyte lysis, blood from healthy human volunteers was washed and labeled with a cocktail of 12 antibodies. Samples were analyzed on a Becton-Dickinson FACSAria TM equipped with three lasers. Data were compared with lineage-specific panels using 5-8 Ab combinations per lineage. Acquired data were analyzed using FlowJo software. Our 12-color panel allows for the identification of the following major subsets of circulating cells in a single tube: CD41 and CD81 T lymphocytes, B lymphocytes, NK cells, NKT cells, monocyte subsets (CD14 and/or CD16), and five nonoverlapping HLA-DR1Lin2 subsets: CD341 hematopoietic stem cells, CD1231 plasmacytoid DC, and three subsets of CD11c1 myeloid DC expressing either CD16, CD1c (BDCA-1), or CD141 (BDCA-3). We have developed a single flow cytometry panel that allows for simultaneous detection of the lymphocyte and monocyte cell populations and all known DC subsets. Studying these major players of the immune system in one single panel may give us a broader view of the immune response during HIV infection and the ability to better define the role of individual cell types in Acquired Immune Deficiency Syndrome (AIDS) pathogenesis. ' 2010 International Society for Advancement of Cytometry

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An 11-color flow cytometric assay for identifying, phenotyping, and assessing endocytic ability of peripheral blood dendritic cell subsets in a single platform

Cited by 26 publications

References 29 publications

Sustained Hyperresponsiveness of Dendritic Cells Is Associated with Spontaneous Resolution of Acute Hepatitis C

Sustained Hyperresponsiveness of Dendritic Cells Is Associated with Spontaneous Resolution of Acute Hepatitis C

Competitive SWIFT cluster templates enhance detection of aging changes

Evaluation of a 12‐color flow cytometry panel to study lymphocyte, monocyte, and dendritic cell subsets in humans

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