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eBioscience™ Human Regulatory T Cell Staining Kit #2 (Invitrogen™)

This Human Regulatory T cell Staining Kit contains all of the buffers and monoclonal antibodies for CD4, CD25, and Foxp3 necessary to successfully stain and identify regulatory T cells from human peripheral blood cells.

The RPA-T4 monoclonal antibody reacts with human CD4, a 59 kDa glycoprotein found on the surface of the majority of thymocytes, a subset of mature T cells (T helper cells), and at lower levels on monocytes. The BC96 monoclonal antibody reacts with human CD25 (also known as interleukin-2 receptor alpha, IL-2R alpha), a 55 kDa surface protein expressed by early progenitors of T cells and B cells, by mature, activated T cells and B cells, and at constitutively high levels on regulatory T cells. The PCH101 monoclonal antibody reacts with the amino terminus of human Foxp3 also known as FORKHEAD BOX P3, SCURFIN, and JM2. Foxp3 is a 49-55 kDa protein and a member of the forkhead/winged-helix family of transcription factors. It was identified as the gene responsible for the X-linked lymphoproliferative disease observed in scurfy (sf) mice and in the human disorder, X-linked autoimmunity-allergic dysregulation syndrome (XLAAD). Constitutive expression of Foxp3 mRNA has been shown in CD4+CD25+ regulatory T cells (Treg), and ectopic expression of Foxp3 in CD4+CD25- cells imparts a Treg phenotype in these cells.

Reactivity/Species
Human

Reported Application
Intracellular Staining Followed by Flow Cytometric Analysis

eBioscience™ Mouse Regulatory T Cell Staining Kit #1 (Invitrogen™)

The FJK-16s antibody reacts with mouse/rat Foxp3 also known as FORKHEAD BOX P3, SCURFIN, and JM2; cross reactivity of this antibody to other proteins has not been determined. Foxp3 a 49-55 kDa protein, is a member of the forkhead/winged-helix family of transcriptional regulators, and was identified as the gene defective in ‘scurfy’ (sf) mice. Constitutive high expression of Foxp3 mRNA has been shown in CD4+CD25+ regulatory T cells (Treg cells), and ectopic expression of Foxp3 in CD4+CD25- cells imparts a Treg phenotype in these cells.

Immunoblotting with FJK-16s antibody has mapped the epitope to amino acids 75-125 of the mouse Foxp3 protein. In the human, this region has been shown to be alternatively spliced at the mRNA level. Both the alternatively-spliced and non-spliced isoforms are present in the CD4+CD25+ subset of lymphocytes. Preliminary RT-PCR experiments have not revealed this alternatively-spliced isoform in mouse splenocytes, suggesting different gene regulation in the mouse and human.

Intracellular staining of mouse splenocytes with FJK-16s using the mouse Foxp3 staining sets and protocol reveals approximately 2% of total splenocytes in the C57Bl/6 strain and approximately 3-5% in the BALB/c mouse strain. Multicolor flow cytometric analysis demonstrates approximately 90% of the CD4+CD25+ cells and 4% of the CD4+CD25- cells staining with FJK-16s. B220+, CD11b+, CD11c+, and Ly6G/Gr-1+ cells do not show significant co-staining with FJK-16s. These data are consistent with a recent report which follows expression of Foxp3 using a GFP knock-in (Fontenot et al, 2005).

FJK-16s cross-reacts with rat Foxp3 This has been demonstrated by intracellular staining of Foxp3 and flow cytometry of rat splenocytes using the same method and reagents as used for mouse tissue. Please note that the CD4 and CD25 antibodies included in this kit only recognize the mouse antigens. For staining rat tissue, please use (CD4 FITC cat. 11-0040 and CD25 APC cat. 17-0390)

The anti-mouse Foxp3 Staining Set has been formulated and optimized for the staining of mouse splenocytes with the FJK-16s monoclonal antibody

Not included:
Isotype controls for anti-CD4 (rat IgG2a, cat. 11-4321) and anti-CD25 (rat IgG1, cat. 17-4301)

Host
Rat

Isotype
IgG2a

Reactivity/Species
Mouse

Reported Application
Intracellular Staining Followed by Flow Cytometric Analysis

eBioscience™ Human Regulatory T Cell Staining Kit (Invitrogen™)

This Human Regulatory T cell Staining Kit contains all of the buffers and monoclonal antibodies for CD4, CD25, and Foxp3 necessary to successfully stain and identify regulatory T cells from human peripheral blood cells.

The RPA-T4 monoclonal antibody reacts with human CD4, a 59 kDa glycoprotein found on the surface of the majority of thymocytes, a subset of mature T cells (T helper cells), and at lower levels on monocytes. The BC96 monoclonal antibody reacts with human CD25 (also known as interleukin-2 receptor alpha, IL-2R alpha), a 55 kDa surface protein expressed by early progenitors of T cells and B cells, by mature, activated T cells and B cells, and at constitutively high levels on regulatory T cells. The PCH101 monoclonal antibody reacts with the amino terminus of human Foxp3 also known as FORKHEAD BOX P3, SCURFIN, and JM2. Foxp3 is a 49-55 kDa protein and a member of the forkhead/winged-helix family of transcription factors. It was identified as the gene responsible for the X-linked lymphoproliferative disease observed in scurfy (sf) mice and in the human disorder, X-linked autoimmunity-allergic dysregulation syndrome (XLAAD). Constitutive expression of Foxp3 mRNA has been shown in CD4+CD25+ regulatory T cells (Treg), and ectopic expression of Foxp3 in CD4+CD25- cells imparts a Treg phenotype in these cells.

Reactivity/Species
Human

Reported Application
Intracellular Staining Followed by Flow Cytometric Analysis

eBioscience™ Mouse Regulatory T Cell Staining Kit #3 (Invitrogen™)

The FJK-16s antibody reacts with mouse/rat Foxp3 also known as FORKHEAD BOX P3, SCURFIN, and JM2; cross reactivity of this antibody to other proteins has not been determined. Foxp3 a 49-55 kDa protein, is a member of the forkhead/winged-helix family of transcriptional regulators, and was identified as the gene defective in ‘scurfy’ (sf) mice. Constitutive high expression of Foxp3 mRNA has been shown in CD4+CD25+ regulatory T cells (Treg cells), and ectopic expression of Foxp3 in CD4+CD25- cells imparts a Treg phenotype in these cells.

Immunoblotting with FJK-16s antibody has mapped the epitope to amino acids 75-125 of the mouse Foxp3 protein. In the human, this region has been shown to be alternatively spliced at the mRNA level. Both the alternatively-spliced and non-spliced isoforms are present in the CD4+CD25+ subset of lymphocytes. Preliminary RT-PCR experiments have not revealed this alternatively-spliced isoform in mouse splenocytes, suggesting different gene regulation in the mouse and human.

Intracellular staining of mouse splenocytes with FJK-16s using the mouse Foxp3 staining sets and protocol reveals approximately 2% of total splenocytes in the C57Bl/6 strain and approximately 3-5% in the BALB/c mouse strain. Multicolor flow cytometric analysis demonstrates approximately 90% of the CD4+CD25+ cells and 4% of the CD4+CD25- cells staining with FJK-16s. B220+, CD11b+, CD11c+, and Ly6G/Gr-1+ cells do not show significant co-staining with FJK-16s. These data are consistent with a recent report which follows expression of Foxp3 using a GFP knock-in (Fontenot et al, 2005).

FJK-16s cross-reacts with rat Foxp3 This has been demonstrated by intracellular staining of Foxp3 and flow cytometry of rat splenocytes using the same method and reagents as used for mouse tissue. Please note that the CD4 and CD25 antibodies included in this kit only recognize the mouse antigens. For staining rat tissue, please use (CD4 FITC cat. 11-0040 and CD25 PE cat. 12-0390)

The anti-mouse Foxp3 Staining Set has been formulated and optimized for the staining of mouse splenocytes with the FJK-16s monoclonal antibody.

Not included:
Isotype controls for anti-CD4 (rat IgG2a, cat. 11-4321) and anti-CD25 (rat IgG1, cat. 12-4301)

Host
Rat

Isotype
IgG2a

Reactivity/Species
Mouse

Reported Application
Intracellular Staining Followed by Flow Cytometric Analysis

eBioscience™ Human Regulatory T Cell Staining Kit #3 (Invitrogen™)

This Human Regulatory T cell Staining Kit contains all of the buffers and monoclonal antibodies for CD4, CD25, and Foxp3 necessary to successfully stain and identify regulatory T cells from human peripheral blood cells.

The RPA-T4 monoclonal antibody reacts with human CD4, a 59 kDa glycoprotein found on the surface of the majority of thymocytes, a subset of mature T cells (T helper cells), and at lower levels on monocytes. The BC96 monoclonal antibody reacts with human CD25 (also known as interleukin-2 receptor alpha, IL-2R alpha), a 55 kDa surface protein expressed by early progenitors of T cells and B cells, by mature, activated T cells and B cells, and at constitutively high levels on regulatory T cells. The PCH101 monoclonal antibody reacts with the amino terminus of human Foxp3 also known as FORKHEAD BOX P3, SCURFIN, and JM2. Foxp3 is a 49-55 kDa protein and a member of the forkhead/winged-helix family of transcription factors. It was identified as the gene responsible for the X-linked lymphoproliferative disease observed in scurfy (sf) mice and in the human disorder, X-linked autoimmunity-allergic dysregulation syndrome (XLAAD). Constitutive expression of Foxp3 mRNA has been shown in CD4+CD25+ regulatory T cells (Treg), and ectopic expression of Foxp3 in CD4+CD25- cells imparts a Treg phenotype in these cells.

Reactivity/Species
Human

Reported Application
Intracellular Staining Followed by Flow Cytometric Analysis

eBioscience™ Mouse Th17 Cytokine Staining Panel (Invitrogen™)

This mouse Th17 cytokine staining panel includes all reagents needed for simultaneous flow cytometric detection of all the major cytokines produced by the Th17 lineage: IL-17A, IL-17F, IL-21 and IL-22. An anti-CD4 antibody that can be used after fixation and permeabilization, as well as IC Fixation and Permeabilization Buffers, is also included.

CD4+ T helper cells are critical mediators of the cellular immune response. For many years, due to cytokine expression patterns, it was thought that CD4+ T helper cells existed as a dichotomy of lineages named Th1 and Th2. However, further investigation revealed that the T helper cell population was not limited to these two subsets. Although it had long been appreciated that IL-17 (also known as IL-17A) production by T cells was required for protection against some pathogens, studies demonstrated that this cytokine was produced by a unique subset of T helper cells. Subsequent reports definitively showed that T cells could differentiate into IL-17-producing cells in vitro and in vivo independent of Th1 or Th2 development, thereby establishing Th17 cells as a unique T helper cell lineage. In addition to IL-17A expression, Th17 cells have been reported to express IL-17F (and heterodimers of Il-17 and Il-17F), IL-21 and IL-22. Functionally, Th17 cells play a role in host defense against extracellular pathogens by mediating the recruitment of neutrophils and macrophages to infected tissues. Moreover, it is becoming evident that aberrant regulation of Th17 cells may play a significant role in the pathogenesis of multiple inflammatory and autoimmune disorders.

Reactivity/Species
Mouse

Reported Application
Intracellular Staining Followed by Flow Cytometric Analysis

eBioscience™ Mouse Hematopoietic Lineage Biotin Panel (Invitrogen™)

The eBioscience Mouse Hematopoietic Lineage Flow Panel contains 5 biotinylated antibodies that can be used to identify, enrich and/or deplete cells committed to the T, B, NK, myeloid and erythroid lineages based on their cell surface antigen expression.

Reactivity/Species
Mouse

Reported Application
Flow Cytometric Analysis

eZKine™ Th1/Th17 Whole Blood Intracellular Cytokine Kit (Invitrogen™)

This eZKine Th1/Th17 Whole Blood Intracellular Cytokine Kit is designed to rapidly identify cytokine producing T lymphocytes of the Th1 and Th17 lineages after stimulation of whole peripheral blood samples. Stimulation of whole blood in the presence of Protein Transport Inhibitor Cocktail (cat. 00-4980, not included) is followed by red blood cell lysis and fixation in a single step and subsequent permeabilization. Samples are then ready to stain with the Th1/Th17 cocktail and concentration-matched Isotype Control cocktail.

Th1 cells are a CD4+ T cell subset that plays an important role in host defense against intracellular bacteria and viruses. Th1 lineage commitment is controlled by the transcription factor, T-bet, and these cells are a primary source of IFN gamma, which stimulates macrophages, lymphocytes, and PMNs in the destruction of bacterial pathogens. IFN gamma also helps foster the development of cytotoxic lymphocytes (CTL & NK cells) that are responsible for the cell-mediated immune response against viruses and tumor cells. Dysregulation of the Th1 response plays a critical role in many inflammatory and autoimmune diseases.

Th17 cells play a key role in barrier defense against extracellular pathogens such as bacteria and fungi and play a significant role in autoimmune disease. They are defined by their expression of the transcription factor ROR gamma t and the inflammatory cytokine IL-17A. Through their activation and subsequent cytokine production, Th17 trigger pro-inflammatory signaling that promotes neutrophil mobilization and the expression of antimicrobial peptides. Some plasticity between the Th1 and Th17 lineages has been reported. Furthermore, a pathogenic Th17 subpopulation has been described with expression of both IL-17 and IFN gamma, as well as ROR gamma t and T-bet. These cells are implicated in several inflammatory and autoimmune diseases.

Reactivity/Species
Human

Reported Application
Intracellular Staining Followed by Flow Cytometric Analysis

eBioscience™ Human/Non-Human Primate Regulatory T Cell Staining Kit #1 (Invitrogen™)

This Human/Non-human Primate Regulatory T cell Staining Kit contains all of the buffers and monoclonal antibodies for CD4, CD25, and Foxp3 necessary to successfully stain and identify regulatory T cells from human, rhesus, cynomolgus, or chimpanzee peripheral blood cells. This kit may also be used to identify regulatory T cells in baboons, using the CD25 and Foxp3 monoclonal antibodies, however, the monoclonal antibody for CD4 that is included in this kit does not crossreact with baboon.

The OKT4 monoclonal antibody reacts with human, rhesus, cynomolgus, and chimpanzee CD4, a 59 kDa glycoprotein found on the surface of the majority of thymocytes, a subset of mature T cells (T helper cells), and at lower levels on monocytes. The BC96 monoclonal antibody reacts with human, rhesus, cynomolgus, chimpanzee, and baboon CD25 (also known as interleukin-2 receptor alpha, IL-2R alpha), a 55 kDa surface protein expressed by early progenitors of T cells and B cells, by mature, activated T cells and B cells, and at constitutively high levels on regulatory T cells. The PCH101 monoclonal antibody reacts with the amino terminus of human, rhesus, cynomolgus, chimpanzee, and baboon Foxp3 also known as FORKHEAD BOX P3, SCURFIN, and JM2. Foxp3 is a 49-55 kDa protein and a member of the forkhead/winged-helix family of transcription factors. It was identified as the gene responsible for the X-linked lymphoproliferative disease observed in scurfy (sf) mice and in the human disorder, X-linked autoimmunity-allergic dysregulation syndrome (XLAAD). Constitutive expression of Foxp3 mRNA has been shown in CD4+CD25+ regulatory T cells (Treg), and ectopic expression of Foxp3 in CD4+CD25- cells imparts a Treg phenotype in these cells.

Reactivity/Species
Human, Monkey

Reported Application
Intracellular Staining Followed by Flow Cytometric Analysis

eBioscience™ Mouse Regulatory T Cell Staining Kit #2 (Invitrogen™)

The FJK-16s antibody reacts with mouse/rat Foxp3 also known as FORKHEAD BOX P3, SCURFIN, and JM2; cross reactivity of this antibody to other proteins has not been determined. Foxp3 a 49-55 kDa protein, is a member of the forkhead/winged-helix family of transcriptional regulators, and was identified as the gene defective in ‘scurfy’ (sf) mice. Constitutive high expression of Foxp3 mRNA has been shown in CD4+CD25+ regulatory T cells (Treg cells), and ectopic expression of Foxp3 in CD4+CD25- cells imparts a Treg phenotype in these cells.

Immunoblotting with FJK-16s antibody has mapped the epitope to amino acids 75-125 of the mouse Foxp3 protein. In the human, this region has been shown to be alternatively spliced at the mRNA level. Both the alternatively-spliced and non-spliced isoforms are present in the CD4+CD25+ subset of lymphocytes. Preliminary RT-PCR experiments have not revealed this alternatively-spliced isoform in mouse splenocytes, suggesting different gene regulation in the mouse and human.

Intracellular staining of mouse splenocytes with FJK-16s using the mouse Foxp3 staining sets and protocol reveals approximately 2% of total splenocytes in the C57Bl/6 strain and approximately 3-5% in the BALB/c mouse strain. Multicolor flow cytometric analysis demonstrates approximately 90% of the CD4+CD25+ cells and 4% of the CD4+CD25- cells staining with FJK-16s. B220+, CD11b+, CD11c+, and Ly6G/Gr-1+ cells do not show significant co-staining with FJK-16s. These data are consistent with a recent report which follows expression of Foxp3 using a GFP knock-in (Fontenot et al, 2005).

FJK-16s cross-reacts with rat Foxp3 This has been demonstrated by intracellular staining of Foxp3 and flow cytometry of rat splenocytes using the same method and reagents as used for mouse tissue. Please note that the CD4 and CD25 antibodies included in this kit only recognize the mouse antigens. For staining rat tissue, please use (CD4 FITC, Cat. No. 11-0040 and CD25 PE, Cat. No. 12-0390)

The anti-mouse Foxp3 Staining Set has been formulated and optimized for the staining of mouse splenocytes with the FJK-16s monoclonal antibody.

Not included:
Isotype controls for anti-CD4 (rat IgG2a, Cat. No. 11-4321) and anti-CD25 (rat IgG1, Cat. No. 12-4301)

Host
Rat

Isotype
IgG2a

Reactivity/Species
Mouse

Reported Application
Intracellular Staining Followed by Flow Cytometric Analysis

eBioscience™ Human Regulatory T Cell Whole Blood Staining Kit (Invitrogen™)

This Human Regulatory T Cell Whole Blood Staining Kit contains the buffers and monoclonal antibody necessary to successfully stain and identify Foxp3 cells in whole blood samples. The PCH101 monoclonal antibody reacts with the amino terminus of human Foxp3 also known as FORKHEAD BOX P3, SCURFIN, and JM2. Foxp3 is a 49-55 kDa protein and a member of the forkhead/winged-helix family of transcription factors. It was identified as the gene responsible for the X-linked lymphoproliferative disease observed in scurfy (sf) mice and in the human disorder, X-linked autoimmunity-allergic dysregulation syndrome (XLAAD). Constitutive expression of Foxp3 mRNA has been shown in CD4+CD25+ regulatory T cells (Treg), and ectopic expression of Foxp3 in CD4+CD25- cells imparts a Treg phenotype in these cells.

The PCH101 antibody crossreacts with rhesus, chimpanzee, and cynomolgus Foxp3 PCH101 recognizes a different epitope of Foxp3 than clones 236A/E7 and 150D/E4.

Host
Rat

Isotype
IgG2a, kappa

Reactivity/Species
Human

Reported Application
Intracellular Staining Followed by Flow Cytometric Analysis

eBioscience™ Human Th17 Cytokine Staining Panel (Invitrogen™)

This human Th17 cytokine staining panel includes all reagents needed for simultaneous flow cytometric detection of all the major cytokines produced by the Th17 lineage: IL-17A, IL-17F, IL-21 and IL-22. An anti-CD4 antibody that can be used after fixation and permeabilization, as well as IC Fixation and Permeabilization Buffers, is also included.

CD4+ T helper cells are critical mediators of the cellular immune response. For many years, due to cytokine expression patterns, it was thought that CD4+ T helper cells existed as a dichotomy of lineages named Th1 and Th2. However, further investigation revealed that the T helper cell population was not limited to these two subsets. Although it had long been appreciated that IL-17 (also known as IL-17A) production by T cells was required for protection against some pathogens, studies demonstrated that this cytokine was produced by a unique subset of T helper cells. Subsequent reports definitively showed that T cells could differentiate into IL-17-producing cells in vitro and in vivo independent of Th1 or Th2 development, thereby establishing Th17 cells as a unique T helper cell lineage. In addition to IL-17A expression, Th17 cells have been reported to express IL-17F (an heterodimers of IL-17A and Il-17F), IL-21 and IL-22. Functionally, Th17 cells play a role in host defense against extracellular pathogens by mediating the recruitment of neutrophils and macrophages to infected tissues. Moreover, it is becoming evident that aberrant regulation of Th17 cells may play a significant role in the pathogenesis of multiple inflammatory and autoimmune disorders.

Reactivity/Species
Human

Reported Application
Intracellular Staining Followed by Flow Cytometric Analysis

eZKine™ 4-Color Compensation Control Kit (Invitrogen™)

This eZKine™ 4-Color Compensation Control Kit is designed for compensation setup with eZKine™ Whole Blood Intracellular Cytokine Kits. It contains the necessary components to create single-color compensation controls for use with eZKine™ cocktails, including OneComp eBeads and 4 vials of antibody representing each of the 4 fluorochromes used in eZKine™ cocktails. OneComp eBeads can be stained with each of the 4 fluorochrome-conjuated antibodies to set up single-color compensation control tubes.

Reactivity/Species
Human

Reported Application
Flow Cytometric Analysis

eZKine™ CD8 Activation 1 Whole Blood Intracellular Cytokine Kit (Invitrogen™)

This eZKine CD8 Activation 1 Whole Blood Intracellular Cytokine Kit is designed to rapidly identify cytokine producing T lymphocytes of the CD8+ T cell lineage after stimulation of whole peripheral blood samples. Stimulation of whole blood in the presence of Protein Transport Inhibitor Cocktail (cat. 00-4980, not included) is followed by red blood cell lysis and fixation in a single step and subsequent permeabilization. Samples are then ready to stain with the CD8 Activation 1 cocktail and concentration-matched Isotype Control cocktail.

CD8+ T cells, or cytotoxic T lymphocytes, are immune cells that are responsible for the detection and clearance of virally or bacterically infected host cells. Upon recognition of antigen displayed on the surface of infected cells, activated CD8+ T cells upregulate markers of early activation such as CD69 and FAS ligand, followed by the release of granules containing granzymes and perforin as well as secretion of cytokines such as IFN gamma and TNF alpha. Granzymes and perforin act in concert to directly lyse infected target cells, while cytokines have paracrine effects including the recruitment and activation of macrophages, lymphocytes, and PMNs. IFN gamma is important for the upregulation of MHC class I and II on nearby cells as well as promoting Th1 cell differentiation.

Reactivity/Species
Human

Reported Application
Intracellular Staining Followed by Flow Cytometric Analysis