Amine reactive dyes: An effective tool to discriminate live and dead cells in polychromatic flow cytometry

Perfetto, Stephen P.; Chattopadhyay, Pratip K.; Lamoreaux, Laurie; Nguyen, Richard; Ambrozak, David R.; Koup, Richard A.; Roederer, Mario

doi:10.1016/j.jim.2006.04.007

Cited by 197 publications

(183 citation statements)

References 10 publications

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“…To this buffer, 50 ml of each mAb dye mix was added plus 5 ml of the amine-reactive viability dye ViViD (Invitrogen) to determine dead cells, with incubation in the dark at 4˚C for 30 min (35). The mAb used for flow cytometry are listed in Supplemental …”

Section: Flow Cytometrymentioning

confidence: 99%

NK Cells Influence Both Innate and Adaptive Immune Responses after Mucosal Immunization with Antigen and Mucosal Adjuvant

Hall

Clare

Dougan

2010

The Journal of Immunology

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Section: Flow Cytometrymentioning

confidence: 99%

NK Cells Influence Both Innate and Adaptive Immune Responses after Mucosal Immunization with Antigen and Mucosal Adjuvant

Hall

Clare

Dougan

2010

The Journal of Immunology

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“…55 Therefore, we analyzed the susceptibility of CM9-specific CD8 ϩ T cells to cell death after stimulation with CM9 peptide for 14 to 16 hours. CM9-specific CD8 ϩ T cells that were negative for Vivid, an amine reactive viability dye, 56 further up-regulated PD-1 upon stimulation with CM9 ( Figure 6A left panel). This up-regulation of PD-1 was associated with extensive sensitivity to cell death, estimated by annexin V positivity (57.4% Ϯ 7.5% annexin V ϩ , n ϭ 5, versus 23.4% Ϯ 5.3% annexin V ϩ , n ϭ 5, for PD-1 high and PD-1 interm CM9 ϩ CD8 ϩ T cells, respectively, P ϭ .006) (Figure 6A left panel).…”

Section: Pd-1 High Siv-specific Cd8 ؉ T Cells Are Characterized By Lomentioning

confidence: 99%

SIV-specific CD8+ T cells express high levels of PD1 and cytokines but have impaired proliferative capacity in acute and chronic SIVmac251 infection

Petrovas¹,

Price

Mattapallil

et al. 2007

Blood

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170

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Programmed death-1 (PD-1) is a critical mediator of virus-specific CD8 ؉ T-cell exhaustion. Here, we examined the expression of PD-1 on simian immunodeficiency virus (SIV)-specific CD8 ؉ T cells and its possible involvement in regulation of cytokine production, proliferation, and survival of these cells. The majority of SIV-specific CD8 ؉ T cells expressed a PD-1 high phenotype, independent of their differentiation status, in all tissues tested. PD-1 expression gradually declined on CD8 ؉ T cells specific for SIV-derived epitopes that had undergone mutational escape, indicating that antigen-specific TCR stimulation is the primary determinant of PD-1 expression. SIV-specific PD-1 high CD8 ؉ T cells produced IFN-␥, TNF-␣, and IL-2 under cognate peptide stimulation. While CD8 ؉ T cells that proliferated in response to antigen had a PD-1 high phenotype, it was determined that there was a reduced proliferative capacity of PD-1 high compared with PD-1 low SIVspecific CD8 ؉ T cells. PD-1 high SIV-specific CD8 ؉ T cells were highly susceptible to IntroductionVirus-specific CD8 ϩ T cells are the predominant effectors through which the immune system controls viral infections. 1,2 While several lines of evidence indicate that human immunodeficiency virus (HIV)-specific CD8 ϩ T cells are involved in the control of HIV replication, 3-10 several reports have focused on intrinsic defects in these cells to explain their failure to clear the virus, thereby leading to progression to acquired immunodeficiency syndrome (AIDS) in all infected individuals. [11][12][13][14] Similarly, simian immunodeficiency virus (SIV)-specific CD8 ϩ T cells contribute substantially to the partial control of viremia in rhesus macaques; depletion of CD8 ϩ T cells results in increased viremia in SIV-infected animals, 15,16 while viral escape at targeted epitopes accelerates disease progression and death in vaccinated animals. 4 Furthermore, in acute SIV infection, cytotoxic SIV-specific CD8 ϩ T cells appear concurrently with the waning of early viremia. 17 As in human infection with HIV, functional defects in SIV-specific CD8 ϩ T cells have been reported, potentially explaining why virtually all SIV-infected rhesus macaques progress to simian AIDS and death despite a readily measurable CD8 ϩ T-cell response. [17][18][19][20] Therefore, understanding factors that regulate the function(s) of SIV-and HIV-specific CD8 ϩ T cells is critical in the fight against AIDS.Chronic viral infection with ongoing antigenic stimulation often results in exhaustion of virus-specific CD8 ϩ T cells. 21,22 Chronically stimulated virus-specific CD8 ϩ T cells express only low levels of receptors for IL-7 and IL-15 23 and lose the ability to maintain homeostatic proliferation. In addition, they lose the ability to produce key cytokines that are critical for the maintenance of CD8 ϩ T-cell memory. 24 In humans, the function and phenotype of chronically stimulated CD8 ϩ T cells differ among viral infections. HIV-specific CD8 ϩ T cells are less polyfunctional and more sensitive ...

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“…Estas células podrían generar fluorescencia no específica y aumentar el ruido de fondo. La adición del canal de descarte es recomendada cuando se evalúan poblaciones con bajas frecuencias y puede ser agregado en conjunto con el marcador de viabilidad (17,32). Perfetto, et al, recomiendan que, para una mejor detección y discriminación de subpoblaciones de LT, se deben incluir en el canal de descarte conjugados para las moléculas CD19 (linfocitos B), CD56 (células NK) y CD14 (monocitos) (5).…”

Section: Discussionunclassified

Diseño de un panel multicolor para evaluar moléculas intracelulares y de superficie mediante citometría de flujo

et al. 2013

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* Los autores contribuyeron de igual manera en el presente estudio.Institución donde se llevó a cabo el trabajo: Pontificia Universidad Javeriana, Bogotá, Colombia.Introducción. La citometría de flujo permite detectar la presencia de moléculas intracelulares y de superficie, de forma simultánea sobre cada célula. Objetivo. Describir un método para la construcción armónica de un panel multicolor con 11 parámetros para el análisis fenotípico y funcional de linfocitos T (LT) CD8 + por citometría de flujo. Materiales y métodos. Para la construcción del panel multicolor, se seleccionaron las moléculas y se titularon los conjugados con fluorocromos para la determinación de CD3, CD8, CCR7, CD28, CD27, CD45RA, CD95 y CD127, en células mononucleares de sangre periférica. Para la evaluación del panel, se hizo la construcción progresiva adicionando uno a uno los conjugados y la fluorescencia menos uno (FMO). Este método fue aplicado para células ex vivo y para evaluar la producción de IFNγ, IL-2 y TNFα frente al estímulo con la enterotoxina B de Staphylococcus aureus (SEB) y al antígeno crudo de Trypanosoma cruzi. Finalmente, se procedió al análisis de las subpoblaciones de LT CD8 + ex vivo en individuos sanos. Resultados. La evaluación de las moléculas con los conjugados no mostró interferencia en las señales de fluorescencia. Las frecuencias de las subpoblaciones de LT CD8 + evaluadas fueron cercanas a los valores reportados en otros estudios. Además, se observó que la frecuencia de LT CD8 + productores de IFNγ, IL-2 y TNFα fue mayor a las seis horas de cultivo con SEB y con el antígeno crudo de T. cruzi. Conclusiones. El método aplicado para la construcción del panel multicolor permite obtener frecuencias de las subpoblaciones de LT CD8 + que corresponden a lo reportado en la literatura científica. Objective: To describe a method for building up a harmonic multicolor panel with 11 flow cytometry parameters for phenotypic and functional analysis on CD8 + T lymphocytes. Materials and methods: For the multicolor panel construction, we selected the molecules and titred conjugated antibodies with fluorochromes for CD3, CD8, CCR7, CD28, CD27, CD45RA, CD95 and CD127 determination in peripheral blood mononuclear cells (PBMC). To evaluate the panel, the conjugated antibodies were gradually added one by one and fluorescence minus one (FMO) test was performed. This method was applied to assess ex vivo subpopulations of T cells and the production of intracellular IFNγ, IL-2 and TNFα using polyclonal stimulation with enterotoxin B from Staphylococcus aureus (SEB) and antigen-specific cells with crude Trypanosoma cruzi antigen. Finally, the ex vivo CD8 + T lymphocyte subpopulations frequencies were analyzed in healthy individuals. Results: The evaluation of the selected molecules and conjugates did not show interference in the fluorescence signals and detection. The frequencies of CD8 + T cells evaluated were similar to the values reported in other studies. Additionally, we observed that the frequency of CD8 + T lymphocytes

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Amine reactive dyes: An effective tool to discriminate live and dead cells in polychromatic flow cytometry

Cited by 197 publications

References 10 publications

NK Cells Influence Both Innate and Adaptive Immune Responses after Mucosal Immunization with Antigen and Mucosal Adjuvant

NK Cells Influence Both Innate and Adaptive Immune Responses after Mucosal Immunization with Antigen and Mucosal Adjuvant

SIV-specific CD8+ T cells express high levels of PD1 and cytokines but have impaired proliferative capacity in acute and chronic SIVmac251 infection

Diseño de un panel multicolor para evaluar moléculas intracelulares y de superficie mediante citometría de flujo

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