Shop All Pathway Analysis Cell Based Assay Kits

CellSensor™ NFKB-bla Jurkat Cell Line

The CellSensor® NFκB-bla Jurkat cell line contains a beta-lactamase reporter gene under control of the Nuclear Factor Kappa B (NFκB) response element stably integrated into Jurkat cells. To construct this cell line, the NFκB-bla construct was transduced into Jurkat cells by lentivirus. Flow cytometry was used to isolate cells responsive to Tumor Necrosis Factor alpha (TNFα). This cell line was validated for DMSO tolerance, cell number, stimulation time, substrate loading time, and Z;-factor and EC50 concentrations for TNF&alpha. The CellSensor® NFκB-bla Jurkat cell line is responsive to TNFα and can be used to probe NFκB signaling pathways, including those involved in apoptosis, viral defense, cancer, inflammation, and autoimmune disease. This cell line can be adapted for high-throughput screening for agonists or antagonists with compound libraries. Candidate drugs can be tested for dose response against this cell line. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ ISRE-bla HEK293T Cell Line

Interferon cell signaling is a critical pathway involved in viral defense and autoimmune disease. The CellSensor® ISRE-bla HEK 293T cell line is responsive to interferon alpha and can be used to probe the JAK/STAT signaling pathway.The CellSensor™ ISRE-bla HEK 293T cell line contains the β-lactamase reporter gene under the control of the interferon stimulated response element (ISRE). This cell line was engineered by transduction of the ISRE-bla construct into HEK 293T cells by lentivirus. Flow cytometry was then used to isolate a pool of clones responsive to interferon alpha a stimulation. A dose response curve for this assay against interferon alpha is available in the validation packet. Academic and non-profit customers, please inquire for special pricing.

LanthaScreen™ STAT3 GripTite™ Cell Line

The JAK/STAT3 signaling pathway is known to be activated by cytokines such as IL-6. In this pathway, binding of IL-6 to its cell-surface receptors results in the activation of JAKs, which in turn phosphoactivate STAT3 proteins at a specific tyrosine residue (Y705). LanthaScreen® STAT3 GripTite™ is a human cell line that constitutively expresses a GFP-STAT3 fusion protein. The JAK/STAT signaling pathway is known to be functionally intact in this cell line; therefore, the GFP-STAT3 fusion protein serves as a substrate for IL-6-inducible phosphorylation. Using this cell line, a lytic immunoassay has been developed in which the phosphorylation state of GFP-STAT3 is detected in cell lysates using a terbium-labeled anti-pY705-STAT3 antibody in a time-resolved FRET (TR-FRET) readout.

The GFP-STAT3 DNA expression construct was transfected into the GripTite™ HEK 293 cell line using Lipofectamine™ 2000 transfection reagent, and the transfected cells were selected with blasticidin. This cell line is a clonal population isolated by flow cytometry using GFP fluorescence as a sorting marker. The assay utilizing this cell line is validated for EC50 and Z’-factors under optimized conditions using IL-6 as a ligand for JAK-mediated GFP-STAT3 phosphorylation. This assay has also been tested under variable experimental conditions, including cell plating density, stimulation time, DMSO tolerance, and assay equilibration time. An alternate "mix-and-read" assay format is also described.

CellSensor™ Gli-bla 22Rv.1 Cell Line

The morphogenic signal Shh provides in the developing CNS induces proliferation of neuronal precursor cells in the developing cerebellum and other tissues. Proliferative signaling by Shh is involved in the development of cancer, including specific brain and skin cancers such as basal cell carcinomas. Signaling takes place through a Patched (PTC-1)⁄Smoothened (SMO) Receptor complex. The activation of Patched by Shh releases the inhibition of Patched on Smoothened leading to Sonic Hedgehog pathway activation of the Gli transcription factor to induce downstream gene expression. The CellSensor® Gli-bla 22Rv1 cell line contains a beta-lactamase reporter gene under control of the Gli response element stably integrated into 22Rv1 cells. This cell line is a clonal population isolated by flow cytometry. The clone was selected based on inhibition of the pathway with KAAD-Cyclopamine. This cell line has also been tested for assay performance under variable conditions, including DMSO concentration, cell number, compound incubation time, and substrate loading time and validated for Z’ under maximal inhibition of β-lactamase activity with Clavulanic Acid.

CellSensor™ irf1-bla CTLL-2 Cell Line

Jak/Stat signaling pathways play essential roles in the cellular responses to distinct cytokines. One of the Jak/Stat pathways, Jak2/Stat5, mediates cell proliferation in response to Interleukin - 3 (IL - 3), prolactin, erythropoietin (Epo), and granulocyte - macrophage colony stimulating factor (GM - CSF). JAK2 gene knockout causes embryonic lethality due to defective erythropoiesis, suggesting that the Jak2/Stat5 pathway plays an important role in red blood cell formation. The recent discovery of an activating mutation in JAK2 (V617F) present in a high percentage of myeloproliferative disease (MPD) patients suggests that the Jak2/Stat5 pathway may be a potential therapeutic target for certain forms of MPD. The activated Stat5 transcription factor recognizes and binds to a specific palindromic DNA sequence found in the promoter region of β - casein, interferon regulatory factor - 1 (irf - 1) and a number of other genes. The CellSensor® irf1 - bla CTLL2 Cell Line contains a beta - lactamase reporter gene under control of the irf - 1 response element stably integrated into CTLL2 cells. CTLL2 cells are a clone of cytotoxic T cells derived from a C57BL/6 mouse, and are cell - growth dependent on mouse Interleukin - 2 (mIL - 2). This cell line is validated for EC50 and Z' - factor under optimized conditions using mIL - 2. This cell line has also been tested under variable assay conditions, including DMSO concentration, cell number, stimulation time, and substrate loading time. Responsiveness to pathway inhibitors was also tested. CellSensor® irf1 - bla CTLL2 cells were stimulated with mIL - 2 in triplicate over the indicated concentration range in a 384 - well format. Cells were incubated for 5 hrs with agonist in 0.5% DMSO and then combined with LiveBLAzer™ - FRET B/G Substrate (CCF4 - AM) for 2.5 hrs. Fluorescence emission values at 460 nm and 530 nm were obtained using a standard fluorescence plate reader. The 460/530 ratios for each replicate plotted against the indicated concentrations of mIL - 2. CellSensor® irf1 - bla CTLL2 cells were treated with Jak Inhibitor 1 over the indicated concentration range in a 384 - well format for 30 min. Cells were then incubated for 5 hrs with mIL - 2 agonist (1 ng/ml) in 0.5% DMSO and then combined with LiveBLAzer™ - FRET B/G Substrate (CCF4 - AM) for 2 hrs. Fluorescence emission values at 460 nm and 530 nm were obtained using a standard fluorescence plate reader, converted to percent inhibition relative to a set of controls (unstimulated cells and EC80 stimulated cells), and plotted against the indicated concentrations of Jak Inhibitor 1. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ AP-1-bla ME-180 Cell Line

The CellSensor® AP-1-bla ME-180 Cell Line contains a beta-lactamase reporter gene under control of the AP-1 response element stably integrated into ME-180 cells. The cell line was created through FACS sorting of cells responsive to stimulation of the AP-1 pathway with Epidermal Growth Factor (EGF). This cell line was validated for DMSO tolerance, stimulant incubation time, substrate loading conditions, Z' and EC50 concentration of Epidermal Growth Factor. The AP-1-bla ME-180 Cell Line responds to agonist treatment as expected from the literature and can be adapted for high-throughput screening for agonists or antagonists of the AP-1 pathway with compound libraries. Candidate drugs can also be tested for dose response against this cell line. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ NFKB-bla Freestyle™ HEK293F Cell Line

Nuclear Factor kappaB (NFκB) signaling regulates genes involved in apoptosis, viral defense, cancer, inflammation, and autoimmune disease. The CellSensor® NFκB-bla FreeStyle™ 293F Cell Line is responsive to tumor necrosis factor alpha (TNFα) and can be used to probe the NFκB signaling pathway. This cell line was validated for DMSO tolerance, incubation time with stimulant and substrate loading conditions. The CellSensor® NFκB-bla FreeStyle™ 293F Cell Line contains a beta-lactamase reporter gene under the control of NFκB response element. The construct was transduced into FreeStyle™ 293F cells by lentivirus. This cell line is a pool isolated for response to TNFα by flow cytometry. It responds to agonist treatment as expected from literature. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ GAS-bla ME-180 Cell Line

The CellSensor® GAS-bla ME-180 cell line contains a beta-lactamase reporter gene under control of the Gamma Activated Sequence (GAS) response element stably integrated into ME-180 cells. To construct this cell line, the GAS-bla construct was transduced into ME-180 cells by lentivirus. Flow cytometry was used to isolate cells responsive to Interferon Gamma (IFN-γ). This cell line is validated for DMSO tolerance, cell number, stimulation time, substrate loading time, as well as Z'and EC50 concentrations for IFN-&gamma. The CellSensor® GAS-bla ME-180 cell line is responsive to IFN-γ and can be used to probe the JAK/STAT signaling pathway. This cell line can be adapted for high-throughput screening of agonist or antagonist compound libraries. Candidate drugs can be tested for dose response against this cell line. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ LEF/TCF-bla SW480 Cell Line

The CellSensor® LEF/TCF-bla SW480 cell line contains a beta-lactamase reporter gene under control of the lymphoid enhancer factor/T cell factor (LEF/TCF) consensus binding sequence stably integrated into SW480 cells, which express a truncated form of the tumor suppressor protein adenomatous polyposis coli (APC). This cell line is a clonal population isolated by flow cytometry, with the pathway validated using RNAi against beta-catenin. The truncated, loss-of-function version of APC expressed in this cell line prevents the proper assembly of the beta-catenin destruction complex. This results in the accumulation of noncomplexed beta-catenin and constitutive activation of this signaling pathway, which is not susceptible to further stimulation with Wnt ligand. This cell line has been tested for assay performance under variable DMSO concentrations and cell numbers. Due to the constitutive activity of this pathway and the absence of known inhibitors, all optimization studies for this CellSensor® line were performed using the beta-lactamase inhibitor clavulonic acid. Treatment with clavulonic acid mimics complete inhibition of the signaling pathway and allows determination of the maximum assay window. Wnt signaling via beta-catenin plays a central role in development and homeostasis. This pathway is invariably disrupted in colorectal tumors and commonly affected by mutation in other cancers. Wnt ligand binding and activation of the frizzled transmembrane receptors transduces the signal to a cytoplasmic protein known as dishevelled, which then inhibits the serine/threonine kinase glycogen synthase kinase 3 beta (GSK3B). This signal leads to functional inactivation and dissociation of a multiprotein beta-catenin destruction complex made up of APC, GSK3B, and a scaffold of axin. This results in dephosphorylation and dissociation of beta-catenin. Unphosphorylated beta-catenin is stabilized and accumulates in the cytoplasm of the cell. Beta-catenin then associates with the LEF/TCF family of transcription factors in the nucleus, leading to transcription and expression of target genes such as c-myc, c-jun, fra, and cyclin D1. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ CYP1A1-bla LS-180 Cell Line

CYP1A1 is an extrahepatic cytochrome P450 with broad substrate specificity involved in the metabolism of numerous potentially toxic and carcinogenic compounds. This cell-based assay measures CYP1A1 induction via the aryl hydrocarbon receptor (AhR) pathway. CYP1A1 induction is regulated by AhR, a transcription factor existing in a multiprotein complex in the cytoplasm. Ligands bind AhR in the cytoplasm of the cells, whereupon the AhR-ligand complex translocates to the nucleus and forms a heterodimer with AhR nuclear translocator (Arnt). This complex then binds the dioxin response element (DRE) in the 5' upstream region of the CYP1A1 promoter, enhancing CYP1A1 expression. The GeneBLAzer® CYP1A1-bla LS-180 cell line contains a beta-lactamase reporter gene whose expression is driven by the CYP1A1 promoter, which serves as a readout for CYP1A1 expression. This cell line has been tested for assay performance under variable conditions, including DMSO concentration, cell number, stimulation time, and substrate loading time. The Z' and EC50 concentrations of 2, 3, 7, 8- tetrachlorodibenzo-p-dioxin (TCDD) were determined. Other CYP1A1 inducers include synthetic and natural chemicals including halogenated and polycyclic aromatic hydrocarbons, and dietary compounds such as flavonoids. Additional testing information using known activators of the pathway are also provided. Academic and non-profit customers, please inquire for special pricing.

LanthaScreen™ IGF-1R GripTite™ Cell Line

The IGF-1R⁄PI3K⁄AKT pathway mediates signals for cell growth, cell survival, transcription, translation, and glucose uptake. IGF-1R is a receptor tyrosine kinase (RTK) that resides at the top of the pathway. Upon growth factor binding extracellularly, this RTK undergoes auto-phosphorylation at multiple intracellular tyrosine residues (resulting in pathway activation). The LanthaScreen® IGF-1R GripTite™ (HEK 293 MSR) cellular assay utilizes a human cell line that constitutively expresses IGF-1R fusion proteins with yellow fluorescent protein (YFP). This kinase target was introduced using lipid transfection and these cells are a clonal population isolated by FACS, using YFP fluorescence as a sorting marker and Blasticidin to maintain cells under selection. Using this cell line, a homogenous immunoassay was developed with a time-resolved FRET (TR-FRET) readout in which the insulin-induced phosphorylation of multiple tyrosine residues on IGF-1R is detected in cell lysates using a generic terbium-labeled phosphospecific antibody (Tb-anti-pY20). This cell line has been validated with different stimuli⁄inhibitors and shows correct EC50 ⁄ IC50 values. Moreover, this assay has been optimized for performance under variable experimental conditions (including cell plating density, agonist stimulation time, DMSO tolerance and assay development time) and displays excellent statistical data (Z' > 0.6) and good signal-to-background.

BacMam ERK2 [pThr185/pTyr187] Cellular Assay Kit (Invitrogen™)

The BacMam ERK2 [pThr185⁄pTyr187] Cellular Assay Kit is a high-throughput screening (HTS) compatible cellular immunoassay for the measuring of phosphorylation of GFP-ERK2. The BacMam ERK2 Cellular Assay Kit can be used with numerous transformed cell lines, primary cells, and stem cells, and allows for the interrogation of ERK2, either as a kinase or GPCR readout.

• Choose cell types that are relevant to your needs
• Get results in weeks instead of months
• Perform Assays on Your Schedule

Use Cell Types Relevant to Your Needs
Understanding signaling in physiologically relevant cell backgrounds is extremely important when predicting the real effects of compounds in humans prior to clinical trials. With the BacMam ERK2 [pThr185⁄pTyr187] Cellular Assay Kit you can choose the appropriate cell type for your research needs, enabling you to design your own experiments, and to reach your research goals faster.

BacMam ERK2 Reagent Compatible Cells
The BacMam reagent included in the kit has been used to successfully transduce numerous transformed cell lines (e.g. U2-OS, HEK 293, and HeLa), primary cells (e.g., fibroblasts, hepatocytes, cardiovascular cells and epithelial cells), and stem cells (e.g. neuronal and mesenchymal) at high transduction efficiency and low levels of cytotoxicity. However, cells of hematopoietic origin show consistently poor transduction by BacMam technology, and are not recommended for use with the current technology.

Develop Assays Faster
The BacMam platform enables transient target expression allowing for reduced time to an HTS ready assay versus traditional stable cell line creation. Traditional stable cell line development requires months of culturing, optimization, and cloning of your cell line. This requires considerable time and resources. With BacMam ERK2 a user can get results in weeks versus months.

Cells Ready When You Are
BacMam Erk2 Reagent is ideal to transduce large quantities of cells in batch mode; transduced cells can be stored frozen in aliquots for later use. This saves you the delays associated with culturing cells. The cells are ready whenever you are. Cells can be assayed typically within hours of thawing, providing assay-ready cells when you need them, with no loss of activity.

For Research Use Only. Not intended for animal or human therapeutic or diagnostic use.

GeneBLAzer™ CHO-K1 Control Kit

The GeneBLAzer® CHO-K1 Control Kit contains constitutively expressing (CMV-bla) CHO-K1 cells and wildtype CHO-K1 cells to provide positive and negative controls for evaluating beta-lactamase activity.

LanthaScreen™ ERK2 A375 Cell Line

The Map Kinase (MapK) signal transduction cascade is activated by growth factors such as EGF, PDGF, and HGF. Binding of these factors to their respective cell surface receptors results in the initiation of receptor tyrosine kinase activity, which leads to the sequential phospho-activation of downstream kinases such as Ras, Raf, MEK, and Erk1⁄2. Activated MEK phosphorylates Erk2 (Mapk1) proteins at a specific Thr-Tyr motif (Thr⁄Tyr 185⁄187). A number of constitutive active kinase mutants within the MAP kinase pathway have been implicated in oncogenesis. One of these mutants is BRAF(V600E), which is the most predominant oncogenic BRAF mutant. The human melanoma cell line A375 endogenously expresses BRAF(V600E), which leads to the constitutive activation of the MAP kinase pathway and phosphorylation of Erk2 in the absence of ligands. LanthaScreenTM Erk2 A375 constitutively expresses GFP-Erk2 under control of a CMV promotor. Using this cell line, a homogenous immuno-assay has been developed in which the phosphorylation state of GFP-Erk2 is detected in cell lysates using a terbium-labeled anti-pTpY-185⁄187-Erk2 antibody, in a time-resolved FRET (TR-FRET) readout. This cell line can be used to evaluate compound activity against BRAF(V600E). GFP-Erk2 lentivirus was transduced into A375 cells, followed by selection with Blasticidin. This cell line is a clonal population isolated by flow cytometry using GFP fluorescence as sorting marker. Using the lytic TR-FRET immuno-assay, this cell line is validated for EC50 and Z’ under optimized conditions using EGF as a ligand for GFP-Erk2 phosphorylation. This assay has also been tested for assay performance under variable experimental conditions, including cell plating density, stimulation time, DMSO tolerance and cell lysis⁄equilibration time. Additional information using alternate stimuli and small molecule inhibitor is also provided.

CellSensor™ CRE-bla CHO K1 Cell Line

The CellSensor® CRE-bla CHO-K1 cell line contains a beta-lactamase reporter gene under control of a cAMP response element (CRE) stably integrated into CHO-K1 cells. This cell line can be used as a parental cell line to build specific G-protein coupled receptor (GPCR) assays after transfection of additional genes of interest or to detect changes in intracellular cAMP levels. CRE-bla CHO-K1 cells have been shown to be responsive to forskolin stimulation. Academic and non-profit customers, please inquire for special pricing.