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Second-Messenger Cyclic AMP Plays an Unusual Role in T Cell Receptor Activation

  • Broad dynamic range—0.06 to 6,000 pmol (5 logs)
  • Flexible—can be used in manual or automated, high-throughput formats
  • Simple—sample dilution not necessary


Our competitive, heterogeneous cAMP-Screen® and cAMP-Screen Direct® immunoassays measure cAMP directly from cell lysates at levels as low as 0.06 to 6,000 pmol, without requiring any sample dilution. Detection is complete typically within 90 minutes, utilizes chemiluminescence, and is amenable for use in automated, high-throughput screening applications.

Cyclic adenosine monophosphate (cAMP) is an important second-messenger in many signal transduction pathways linking activation of cell surface membrane receptors to intracellular responses and, ultimately, to changes in gene expression. The second-messenger cAMP plays a rather complicated role in T cell receptor activation. T cell receptor activation stimulates cAMP production; however, cAMP as part of a negative feedback loop inhibits T cell function and proliferation (Mol Cell Biol 30:1660–1672 (2010)).

The sensitivity and simplicity of the cAMP-Screen Direct® assay provides a powerful tool to measure small changes in cAMP levels, important in the elucidation of the role of cAMP in T cell receptor activation.

cAMP-Screen assay


Figure 1. cAMP-Screen® assay.

For research use only. Not intended for any animal or human therapeutic or diagnostic use.

Molecular Probes® Calcium Indicators for Studying T Cell Activation

  • Widest selection of Ca2+ fluorescent indicators over the range of <50 nM to >50 µM
  • Fixable and permeable dyes, in all detectable colors
  • Adaptable to many platforms, including traditional imaging, high-content screening, and flow cytometry


In one activation pathway, cell surface ligand binding to T lymphocytes results in the hydrolysis of membrane phosphoinositides, and the hydrolytic products, IP3 and DAG, act as second-messengers to mobilize Ca2+ from both intracellular and extracellular stores. This increase in free cytoplasmic Ca2+ is important for the activation of PKC, which in turn activates IL-2, instrumental in the activation and proliferation of T cells. To measure the increase in intracellular Ca2+, we offer various Molecular Probes® indicators that cover a wide dynamic and spectral range.

Ion-sensitive probes have enabled researchers to investigate changes in intracellular free Ca2+ concentrations using fluorescence microscopy, flow cytometry, and fluorescence spectroscopy. The properties and applications of these fluorescent indicators—most of which are derivatives of the Ca2+ chelators EGTA, APTRA, and BAPTA—have been extensively reviewed in the literature.

A few of these molecules are useful to monitor real-time increases in free cytoplasmic Ca2+ in live cells, and many of these indicators are also fixable, for flexibility in detecting Ca2+ increases at different stages. These advantages make cytoplasmic Ca2+ indicators valuable tools for the study of the T cell pathway and T cell activation.

A live bovine pulmonary artery endothelial cell

Figure 2. A live bovine pulmonary artery endothelial cell stained with 500 nM X-rhod-1 AM for 30 minutes at 30°C.

The image was deconvolved using Huygens software (Scientific Volume Imaging, www.svi.nl).
For research use only. Not intended for any animal or human therapeutic or diagnostic use.

Fluorogenic Substrate for the Detection of Activated Caspases

  • DEVD substrate, specific for caspase-3/7 with absorption/emission maxima of ~502/530 nm
  • Simple, no-wash protocol preserves delicate apoptotic cells
  • Suitable for live-cell fluorescence imaging, enabling continuous monitoring of dynamic events
  • Compatible with formaldehyde-based fixation methods, facilitating convenient processing for endpoint assays


During development in the thymus, T cells that fail to produce a functional T cell receptor or that fail to bind self MHC class I molecules with the correct affinity are destroyed by apoptosis. Although mechanisms vary, cysteine aspartic acid proteases, or caspases, play a key role in initiating and executing apoptosis, making the activation of caspases one of the more definitive markers of programmed cell death.

The CellEvent™ Caspase-3/7 Green Detection Reagent comprises a DEVD peptide sequence conjugated to a nucleic acid–binding dye. In the presence of activated caspases-3 and -7, the CellEvent™ reagent fluoresces bright green (monitored with a standard FITC filter set, see figure). This robust assay enables researchers to examine caspase-3 and -7 activation in live cells. Furthermore, since wash steps are not necessary for detection, fragile apoptotic cells typically lost during wash steps are retained. Importantly, the fluorescent signal from CellEvent™ Caspase-3/7 Detection Reagent can survive fixation and permeabilization. This allows flexibility to perform endpoint assays and to probe the sample for other proteins of interest using immunocytochemistry.

Oxidative Stress and Apoptosis

Figure 3. Multiplex measurements of oxidative stress and apoptosis.
HepG2 cells were either treated with a DMSO control (A) or with 50 µM nefazodone (B) for 24 hours. During the last 30 minutes of this treatment, the cells were stained with 5 µM CellROX™ Deep Red Reagent, Hoechst 33342, and 5 µM CellEvent™ Caspase-3/7 Green Detection Reagent. Cells were analyzed on a Thermo Fisher Cellomics ArrayScan® VTI. Cells not treated with nefazodone (A) show very little signal from either the oxidative stress or apoptosis detection reagent, as opposed to the treated cells (B).
For research use only. Not intended for any animal or human therapeutic or diagnostic use.

How Does FOXP3 Regulate T Cells in Adaptive Immunity?

  • ABfinity™ FOXP3 rabbit recombinant monoclonal antibody for sensitive detection
  • Consistent performance for your assays
  • Consistent product from lot to lot


ABfinity™ recombinant rabbit monoclonal antibodies are produced from specific recombinant clones, so antibody performance is consistent over time. ABfinity™ antibodies are available for many targets, including FOXP3 (gene ID #50943). These FOXP3 antibodies have been validated in immunocytochemistry (Cat. No. 700020) and western blotting (Cat. No. 700914).

 

Forkhead box-O transcription factors control expression of immune system genes in regulatory T cells. FOXP3 induces immunosuppressive functions in regulatory T cells and is a potential therapeutic target in tumor cells. ABfinity™ recombinant monoclonal antibodies are ideal tools for signaling pathway investigations. The ABfinity™ technology helps ensure consistent antibody performance, so you don’t have to reoptimize your assay with each new lot.

Immunocytochemistry of HeLa cells labeled with rabbit anti-FOXP3

Figure 4. Immunocytochemistry of HeLa cells labeled with rabbit anti-FOXP3.
Alexa Fluor® 488 goat anti-rabbit  was used at 1:1000 dilution as secondary antibody. Top, left to right: DAPI only (blue), Alexa Fluor® 488 signal only (green); bottom, left to right: Alexa Fluor® 594 phalloidin (red) and the composite image.
For research use only. Not intended for any animal or human therapeutic or diagnostic use.
ABfinity™ Ordering Information

Product
Application*
Species
Qty.
Cat. No.
FOXP3 ABfinity™ Recombinant Rabbit Monoclonal Antibody - Purified
ICC
Human
100 µg
700020
FOXP3 ABfinity™ Recombinant Rabbit Monoclonal Antibody - PurifiedWB
Human
100 µg
700914
*ICC=Immunocytochemistry; WB=Western Blot

Alexa Fluor® Secondary Antibodies Light Up Your Pathway

  • Brighter
  • More photostable
  • Fluorescent over a broader pH range than other dyes


Life Technologies provides an extensive selection of Molecular Probes® secondary immunoreagents for use in fluorescence microscopy. A wide variety of Alexa Fluor® secondary antibodies are available, with a variety of host and target species and a color selection that covers the spectrum from Alexa Fluor® 350 to Alexa Fluor® 750.

 

Stimulation of the T cell receptor pathway is triggered by major histocompatibility complex (MHC) molecules on antigen-presenting cells; this stimulation induces a series of intracellular signaling cascades that ultimately result in cellular proliferation, differentiation, cytokine production, and/or activation-induced cell death.

Immunocytochemistry using specific primary antibodies detected with Alexa Fluor® secondary antibodies is a powerful research tool (see figure). Because of their superior brightness and photostability, the Alexa Fluor® conjugates offer significant advantages for fluorescence-based immunoassays.

Immunocytochemistry of Jurkat cells

Figure 5. Immunocytochemistry of Jurkat cells labeled with rabbit anti–ZAP-70 [pY315/pY319].
Jurkat cells were either untreated (A) or treated with H2O2 (B, C, D) and labeled with rabbit anti–ZAP-70 [pY315/pY319] (2 µg/mL). Alexa Fluor® 488 goat anti-rabbit at 1:1000 dilution was used as secondary antibody. Preincubation of treated cells with the phosphopeptide immunogen decreased signal (D), while preincubation with the nonphosphopeptide did not (C). Blue = Hoechst nuclear stain; green = Alexa Fluor® 488.
For research use only. Not intended for any animal or human therapeutic or diagnostic use.

Measuring IL-2 Secretion After T Cell Activation

  • Accurate measurement of IL-2 in serum, plasma, or supernatant
  • Consistent and reliable results (<10% CV)
  • Incubation time typically only 3 hours


IL-2, also known as T cell growth factor, is a cytokine released by activated CD4+ T cells. It acts in an autocrine manner to promote T cell and NK cell growth. IL-2 is also a T cell differentiation factor, able to induce the production of other lymphokines such as IL-4 and interferon- y. Low concentrations of IL-2 can be detected in normal serum samples or in culture supernatant after mitogen activation of mononuclear cells (PBMCs).

 

T cell receptors are found on the surface of T lymphocytes. When an antigen or mitogen binds to these receptors and activates the mature T lymphocyte, transcription and release of synthesized IL-2 occurs.

ELISA kits allow for quantitative measurement of IL-2 protein. The assay is easy to perform, and results are obtainable within half a day. The assay is sensitive and provides high precision (<10% CV). Invitrogen offers IL-2 assay kits in multiple formats, whether you want to measure IL-2 secretion only or would like to measure IL-2 along with other cytokines in one well using Invitrogen™ Luminex® multiplexing kits.

Representative Standard Curve for the Hu IL-2 ELISA Kit
 
Figure 6. Representative Standard Curve for the Hu IL-2 ELISA Kit. The dynamic range of the assay is 15.6 to 1000 pg/mL.
For research use only. Not intended for any animal or human therapeutic or diagnostic use.
Ordering Information

Product
Qty.
Cat. No.
Human IL-2 ELISA Kit
96 tests
KHC0021
Mouse IL-2 ELISA Kit
96 testsKMC0021
Rat IL-2 ELISA Kit
96 testsKRC0021
Monkey IL-2 ELISA Kit
96 testsKPC0021

T Cell Cytokine Signaling—Multiplex Luminex® Assays

  • Fast and easy cytokine assay protocols—perform and analyze your data in less than one day
  • Superior performance—accurate, reproducible, and sensitive quantitation of multiple cytokines
  • High quality—antibody-based assays with excellent specificity and sensitivity


To facilitate the analysis of a broad range of important cytokines, we have developed a series of multiplex immunoassays for the Luminex® platform that permit the simultaneous detection of numerous important biomarkers—delivering potential savings in cost, time, and sample.

 

The functional aspects of many T cells are tied to the release of cytokines. Cytokines play an important role in T cell activation and are potent T cell growth factors, and many cytokines are secreted by helper T cells. T cell subsets have also been found to produce cytokines differentially in response to diverse stimuli.

Luminex® multiplex assays, in conjunction with the Luminex® 100™, Luminex® 200™, or FLEXMAP 3D® System, are easy to perform and deliver reproducible analytical results, making them valuable research tools in the quantitation of key cytokines.

  • Learn More About Luminex® Assays
  • Learn More About Luminex® 100™ or 200™ System and the FLEXMAP 3D® System
Consistent quantitation

Figure 7. Consistent quantitation of GM-CSF, IFN-γ, IL-1β, IL-2, IL-6, IL-8, IL-10, and TNF-α on both polystyrene (Poly) and magnetic (Mag) bead assays.
Natural samples were tested undiluted, 2-fold diluted, or 5-fold diluted in assay buffer.
For research use only. Not intended for any animal or human therapeutic or diagnostic use.
© 2011 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.  Luminex 200 and FlexMap 3D are trademarks of Luminex Corporation.