BioPath Online™

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

New TNF pathway web page Empower your research today using Invitrogen’s comprehensive portfolio of products and services to investigate the TNF 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 TNF pathway–associated reagents at www.invitrogen.com/TNF.

The TNF-induced (tumor necrosis factor) model of apoptotsis is initiated by the binding of TNF to TNF-R1. This binding initiates a signaling pathway leading to caspase activation via TNF receptor-associated death domain (TRADD) and Fas-associated death domain protein (FADD).  The link between TNF and apoptosis shows why an abnormal production of TNF plays a fundamental role in several human diseases, especially in autoimmune diseases.

The terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) assay is based on the incorporation of modified dUTPs by the enzyme terminal deoxynuclotidyl transferase (TdT) at the 3’-OH ends of damaged DNA, a hallmark as well as the ultimate determinate of apoptosis. Often at this final stage of apoptosis, adherent cells are known to detach or “pop” off.  The new Click-iT® TUNEL Imaging Assays utilize a dUTP modified with an alkyne, a small, bio-orthogonal functional group that enables the nucleotide to be more readily incorporated by TdT than other modified nucleotides, including fluorescein-dUTP (Figure 1).  Detection is based on a click reaction, a copper (I) catalyzed reaction between an azide and alkyne that is complete in 30 minutes.  The small size of the Alexa Fluor® azide detection reagent (MW <~1000) compared to that of an antibody (MW ~150,000) or streptavidin (~60,000) enables facile penetration of complex samples with only mild fixation and permeabilization required.  As a result, the Click-iT® TUNEL imaging assays are fast and complete within 2 hours and deliver accurate consistent performance with content rich results (Figure 2). 

		Figure 1:  TUNEL assay timecourse comparison - % positives detected. HeLa cells were treated with 0.5 µM staurosporine for the time points indicated.  Following fixation and permeabilization, TUNEL assays using either Click-iT® EdUTP or fluorescein dUTP (Promega’s Dead End Kit) were performed according to the manufacturer’s instructions. The % positives were calculated based upon the corresponding negative control. Imaging and analysis was performed using a ThermoFisher Cellomics ArrayScan II.
		Figure 2.  Imaging DNA fragmentation and caspase-3 activity in HeLa with Click-iT® TUNEL Assays. DNA strand breaks typical of late stage apoptosis in HeLa cells visualized using the Click-iT(R) TUNEL Imaging Assay. HeLa cells were treated with 0.5 uM staurosporine for 4 hr, then fixed and permeabilized. The Click-iT® TUNEL Alexa Fluor® 647 Imaging Assay (Cat. no. C10247) was performed. Activated caspase-3 was detected with a polyclonal rabbit primary antibody for cleaved caspase-3 and labeled with Alexa Fluor® 488 goat anti–rabbit IgG (Cat. no. A11008, green fluorescence). Tubulin was detected with a mouse monoclonal anti-tubulin antibody and labeled with Alexa Fluor® 555 goat anti–mouse IgG (Cat. no. A21422, orange fluorescence). Nuclei were stained with Hoechst 33342 (Cat. no. H1399, blue fluorescence). The coverslips were mounted in ProLong® Gold antifade reagent (Cat. no. P36930) and incubated overnight at 4°C before imaging. The cells on the left not only have a high level of caspase-3 activity and DNA strand breaks, but also show a loss of structural integrity consistent with cells undergoing apoptosis.

• Learn more about Click-iT® TUNEL Assays
  

Now available is a convenient Apoptosis Detection Kit (Cat. no. KHM4011) to localize both cytochrome c and complex V proteins.  The kit comes with anti-cytochrome c monoclonal antibody (IgG_2a isotype) and a goat anti-mouse IgG_2a-FITC secondary antibody enabling the researchers to observe the location of cytochrome c in cells by fluorescence microscopy.  This kit also contains an anti-Complex V subunit α (CVα) monoclonal antibody (IgG_2b isotype) and a goat anti-mouse IgG_2b-TXRD secondary antibody. Complex V is an inner mitochondrial membrane protein and serves as a mitochondrial marker - unlike cytochrome c, it is not released into the cytoplasm during apoptosis. Both primary antibodies are reactive in human, mouse and rat cells.

One of the crucial players in apoptosis is cytochrome c, which, under apoptotic conditions, is released from the intermembrane space of the mitochondria to the cytosol.  Once in the cytosol, cytochrome c combines with an adaptor subunit APAF-1 in the presence of dATP, leading to dimerization of APAF-1 and activation of a cysteine protease, caspase 9.  Caspase 9 triggers activation of other caspases, which, in turn, selectively destroy certain proteins such as DNA replicating proteins and cytoskeletal proteins resulting in cell death.  Release of cytochrome c from mitochondria is an early marker of apoptosis.

				Figure 1.  Staurosporine-treated HeLa cells analyzed with anti-cytochrome c antibody. The white arrow indicates cytochrome c release from mitochondria.
				Figure 2.  Staurosporine-treated HeLa cells analyzed with anti-Complex Vα antibody.
				Figure 3.  Figure 1 and 2 merged.

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Two complementary cellular approaches to analyze the NFκB pathway activation by tumor necrosis factor (TNF) family cytokines are now validated for use in high-throughput screening (HTS) applications.

LanthaScreen™ Cellular Assays provide a proximal readout to analyze the phosphorylation and/or ubquitination status of IκB proteins in living cells.  This system utilizes cell lines that stably express GFP- IκB fusion proteins. The phosphorylation and ubquitination states of GFP-IκB is then analyzed in cell lysates using a terbium (Tb)-labeled phospho-specific IκB antibody and poly-ubquitination-specific antibody, respectively, in a time-resolved Föerster-resonance energy transfer (TR-FRET)-based readout. 

CellSensor® Cell Lines monitor the transcriptional function of NFκB using a beta-lactamase reporter gene assay in physiological and disease-relevant cell backgrounds.  Beta-lactamase provides advantages over luciferase and beta-galactosidase reporter enzymes in that it can be detected in living cells with its FRET-based membrane-permeable substrate. The dual wavelength readout allows for ratiometric analysis which significantly reduces experimental variables. 

Both LanthaScreen™ Cellular Assays and CellSensor® Cell Lines were validated by modulating the TNFα/NFκB pathway activation using a known small molecule inhibitor.  The results suggest that these two assays work as integrated HTS-compatible tools to analyze IKK-proximal activity and IKK-mediated downstream pathway function in a physiological context.

Measurement of TNFα-induced IκB phosphorylation and ubiquitination using the Lanthascreen™ IκB alpha Cellular Assay. (Left panel) LanthaScreen™ IκB alpha GripTite cells (K1681) were plated in assay medium for 16 hours before treatment with indicated concentrations of TNFα (PHC3011) for 30 minutes. Cell media were then aspirated and cells lysed in the presence of the following LanthaScreen™ detection antibodies: LanthaScreen™ Tb-anti-IκB alpha [pSer32] (PV3562) or LanthaScreen™ Tb-anti-ubiquitin FK2 (PV4752). The TR-FRET response ratios were plotted against indicated concentrations of TNFα. Error bars represent the standard error of n = 4 or n = 8 values. A concentration-dependent activation of GFP-IκB alpha phosphorylation and ubiquitination is observed using this readout, indicating that this LanthaScreen™ assay can be used for multiple posttranslational modifications. (Right Panel) Cells were treated as described above, except for the preincubation of IKK inhibitor IV prior to stimulation with 1 ng/mL TNFα. This result indicates that both phosphorylation and ubiquitination events can be inhibited at an early step in the NFκB signaling cascade.

Measurement of the TNFα-induced transcriptional activity of NFκB using the CellSensor® NFκB-bla ME-180 reporter assay. (Left Panel) NFκB-bla ME180 cells (12,000 cells/well) cells (K1667) were plated in a 384-well plate and stimulated with TNFα 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 Fluorescence emission values at 460 nm and 530 nm were obtained using a standard fluorescence plate reader and the Response Ratios plotted for the indicated concentrations of TNFα (n=16 for each data point). (Right Panel) Cells were plated as described above and pretreated with indicated concentrations of IKK Inhibitor IV for 45 minutes before the stimulation with TNFα. (n=4 for each data point).

• Learn more about LanthaScreen™ Cellular Assays