Biopath Online T-cell signaling Pathway

Here's what's in this issue:

New pathway web pages
Whether your T cell receptor signaling pathway research involves basic research tools, cell-based assays, or comprehensive screening services, Invitrogen has solutions for you. See our new T cell receptor signaling pathway web page for more information.

T cell receptor signaling pathway
 New T cell receptor signaling pathway web page
Empower your research today using Invitrogen’s comprehensive portfolio of products and services to investigate the T cell receptor signaling pathway—everything from high-quality reagents for basic research and assay development to validated biochemical and cell-based assays, and world-class profiling and screening services.

See our portfolio of T cell receptor signaling pathway–associated reagents at

A number of signaling cascades are mediated through activating the T cell receptor (TCR), ultimately determining cell fate by regulating cytokine production, cell survival, proliferation, and differentiation.

Downstream of the initial activation, the MAPK/ERK pathway is activated by the second messengers diacylglycerol (DAG) and inositol trisphosphate (IP3), which in turn promote activation of transcription factors including NFκB, c-Jun, and c-Fos.  Rapid, quantifiable measurements of phosphorylation events in this pathway are now easier than ever with immunoassays in both single and multiplex formats.

Invitrogen immunoassay kits allow you to quantify protein levels in cell or tissue lysate in less than half a day using 96-well plates.  Furthermore, the assays provide excellent sensitivity for low-expression proteins or small sample volumes. 

Invitrogen phosphoELISA™ kits for both phosphorylation site-specific proteins and total proteins elucidate specific target proteins in the T cell signaling pathway.  These targets are also available for multiplexing studies using the Luminex® platform. 

High sensitivity
 Figure 1. High sensitivity. Data show the result of normalizing data obtained with p38 MAPK [pTpY180/182] phosphoELISA™ (Cat. no. KHO0061) to data obtained with the p38 MAPK Total phosphoELISA™ (Cat. no. KHO0071). Anisomycin treatment increases the level of phosphorylation of  p38 MAPK at threonine 180 and tyrosine 182. This phosphorylation event was detected with as little as 3,000 cells.

Western signal decreases proportionately to ELISA signal in response to decreasing PMA dose

Figure 2. Jurkat cells were treated with varying concentrations of PMA, then tested for ERK1/2 [pTpY185/187] (Cat. no. KHO0091) and total (Cat. no. KHO0081) protein by either phosphoELISA™ or Western blot. Data show that Western signal decreases proportionately to ELISA signal in response to decreasing PMA dose.

Please visit to see the complete menu.
T cells can be distinguished from other lymphocyte types, such as B cells and natural killer cells, by the presence of a special receptor on their surfaces called a T cell receptors (TCR). T cells can be further categorized into distinct subsets by specific cell surface markers.

There are a number of techniques available for determination of a T cell population in a given sample; however, flow cytometry is one of the fastest and most reliable.

To match the ever-expanding detection capabilities available in new instrumentation, Molecular Probes® Qdot® nanocrystals offer an exciting new array of fluorescent labels for flow cytometry (see figure 1). Qdot® nanocrystals are fluorescent labels that can be excited with UV or violet light sources, as well as with longer-wavelength light sources, and exhibit long Stokes shifts and relatively narrow emission peaks. The result is greater flexibility and sensitivity in designing multicolor flow cytometry panels.

T-cell signaling pathway
Figure 1. Antigen detection in human peripheral blood lymphocytes. Cells were incubated with mouse anti–human CD27 antibody labeled with Qdot® 655 nanocrystals (A) or mouse anti–human CD45 antibody labeled with Qdot® 705 nanocrystals (B) and analyzed using a BD™ LSR II flow cytometer. The gray overlay peaks represent data from unstained cells.


LanthaScreen™ kinase activity assays have been developed and validated for several key kinases in the T cell receptor signaling pathway. With LanthaScreen™ assays, you can rapidly apply TR-FRET technology to detect kinase activity in a simple and purified system where kinase, fluorescein-labeled substrate, and ATP are allowed to react. Then EDTA (to stop the reaction) and terbium-labeled antibody (to detect phosphorylated product) are added.

The antibody binds to the phosphorylated fluorescein-labeled substrate, resulting in an increased TR-FRET value. This value is the ratio of the acceptor (fluorescein) signal to the donor (terbium) signal. The amount of antibody bound to the tracer is directly proportional to the amount of phosphorylated substrate present; therefore, kinase activity can be detected and measured by an increase in TR-FRET value.

Several beta-lactamase reporter gene assays (CellSensor® assays) also monitor the transcriptional function of various transcription factors downstream of T cell receptor (TCR) activation. Beta-lactamase’s advantages over luciferase and beta-galactosidase reporter enzymes lie in its ability to be detected in living cells with its FRET-based membrane-permeable substrate. The dual wavelength readout allows ratiometric analysis, which significantly reduces experimental variables. Our results suggest that these assays can analyze TCR-mediated downstream pathway functions in a high-throughput manner. 

T-cell signaling pathway
Measurement of tyrosine kinase (ZAP70, LCK, and ITK) activity using Lanthascreen™ technology. Protein tyrosine kinases within the T cell receptor signaling pathway can be assayed using the LanthaScreen™ toolbox of reagents.  A two-fold serial dilution of kinase (ZAP70 (P2782); LCK (P3043), or ITK (P3875)), were incubated with 200 nM Fluorescein-poly GT (PV3610) and 2 mM ATP in a 384-well plate.  A 2X solution of 20 mM EDTA and 4 nM LanthaScreen™ Tb-PY20 Antibody (PV3552) stopped the reaction and developed the TR-FRET signal.  Time resolved fluorescence emission values at 520 nm and 490 nm were collected on a BMG LABTECH Pherastar plate reader using standard TR-FRET settings.
T-cell signaling pathwayMeasurement of TCR-mediated transcriptional activity of NFAT using the CellSensor® NFAT-bla Jurkat reporter assay. NFAT-bla Jurkat cells (K1671) (25,000 cells/well) were plated in a 384-well format and stimulated with anti-CD3/anti-CD28 (BD Pharmingen/555329 and 555725) plus cross-linker (Pierce/31160) over the indicated concentration range in the presence of 0.1% DMSO for 5 hours. Cells were then loaded with LiveBLAzer™-FRET B/G Substrate for 2 hours. Fluorescence emission values at 460 nm and 530 nm were obtained using a standard fluorescence plate reader and were plotted for the indicated concentrations of antibodies (n=16 for each data point).