Shop All Pathway Analysis Cell Based Assay Kits

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™ SIE-bla THP-1 Cell Line

The CellSensor® SIE-bla THP-1 cell line contains a beta-lactamase reporter gene under control of the Sis Inducible Element (SIE), stably integrated into THP-1 cells. To construct this cell line, the SIE-bla construct was transduced into THP-1 cells by lentivirus. Flow cytometry was used to isolate cells responsive to Interferon Gamma (IFNγ). 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® SIE-bla THP-1 cell line is responsive to IFNγ and can be used to analyze the JAK/STAT pathway using the GeneBLAzer® Loading Substrates. Academic and non-profit customers, please inquire for special pricing.

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.

CellSensor™ TrkA-NFAT-bla CHO-K1 Cell Line

Neurotrophins (NGF, BDNF, NT-3, and NT-4) and their transmembrane receptors (TrkA, TrkB, TrkC, and P75NTR) play important roles in the regulation of neuronal and non-neuronal cell proliferation, differentiation, survival, and death. Neurotrophin signaling also mediates neuronal higher-order activities, such as learning, memory, and behavior. Alterations in neurotrophin levels and their receptors have been implicated in neurodegenerative disorders such as Alzheimer's disease and Huntington's disease, as well as psychiatric disorders. Neurotrophins propagate their signal by activating multiple signaling pathways. One of the signaling pathways of NGF (the ligand for TrkA) activates phospholipase C, releasing DAG and IP3, increasing downstream intracellular calcium, and activating protein kinase C. This promotes the translocation of the transcription factor, nuclear factor of activated T-cells (NFAT), from the cytosol into the nucleus, resulting in NFAT-dependent transcription.

The CellSensor® TrkA-NFAT-bla CHO-K1 Cell Line was engineered by integrating the human TrkA expression plasmid into the genome of existing CellSensor® NFAT-bla CHO-K1 Cell Line, which is engineered to express beta-lactamase under the control of NFAT. This cell line has been tested for assay performance under variable conditions, including DMSO concentration, cell number, stimulation time, substrate loading time, and in cryopreserved cells, and has been validated for Z´-factor and EC50 values under optimized conditions using NGF 2.5s.

Additional testing information using various small molecule inhibitors is also provided.

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™ NFAT-bla Jurkat Cell Line

The CellSensor® NFAT-bla Jurkat cell line contains a beta-lactamase reporter gene under control of a Nuclear Factor of Activated T Cells (NFAT) response element stably integrated into Jurkat 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. NFAT-bla Jurkat cells have been shown to be responsive to phorbol 12-myristate 13-acetate (PMA) and Ionomycin stimulation. Additional testing data using alternate stimuli is available upon request. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ HRE-bla HCT-116 Cell Line

Hypoxia is an almost universal hallmark of solid tumors. Adaptation to hypoxia is critical for tumor survival and growth, and is mediated largely by transcriptional activation of genes that facilitate short - (e.g., glucose transport) and long - term (e.g., angiogenesis) adaptive mechanisms. This coordinated homeostatic response is mediated in large part through the activation of the heterodimeric transcription factor hypoxia - inducible factor 1 (HIF - 1). Under conditions of normal oxygenation, the regulated HIF - 1α is hydroxylated and degraded by the proteasome system. As oxygen becomes rate limiting, hydroxylation diminishes and HIF - 1α accumulates and heterodimerizes with the constitutively present α - subunit. The binding of this complex to the cognate hypoxia - response element (HRE) results in transcriptional activation of genes containing such elements within promoter or enhancer elements. The CellSensor® HRE-bla HCT - 116 Cell Line contains a beta - lactamase reporter gene under control of the HRE stably integrated into HCT - 116 cells. This cell line is validated for EC50 and Z'; - factor under optimized conditions using deferoxamine (DFO) and Cobalt Chloride. This cell line has also been tested under various experimental conditions, including DMSO concentration, cell number, stimulation time, and substrate loading time. CellSensor ® HRE-bla HCT - 116 cells were stimulated with deferoxamine (DFO) over the indicated concentration range in a 384 - well format. Cells were incubated for 24 hrs with agonist and 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. The Response Ratios were plotted against the indicated concentrations of DFO. CellSensor® HRE - bla HCT - 116 cells were stimulated with Cobalt Chloride (CoCl2)over the indicated concentration range in a 384 - well format. Cells were incubated for 24 hrs with agonist and 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. Response ratios were plotted against the indicated concentrations of CoCl2. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ irf1-bla Ba/F3 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 BA/F3 Cell Line contains a beta - lactamase reporter gene under control of the irf - 1 response element stably integrated into BA/F3 cells. This cell line is validated for EC50 and Z' - factor under optimized conditions using mouse IL - 3 (mIL - 3). This cell line has also been tested under variable experimental conditions, including DMSO concentration, cell number, stimulation time, and substrate loading time. Responsiveness to Jak Inhibitor 1, a small molecule inhibitor, was also tested. CellSensor® irf1 - bla BA/F3 cells were treated with mIL - 3 in triplicate over the indicated concentration range in a 384 - well format. Cells were incubated for 5 hrs with mIL - 3 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 Response Ratios for each replicate were plotted against the indicated concentrations of mIL - 3. CellSensor ® irf1 - bla BA/F3 cells were treated with Jak Inhibitor 1 for 30 min over the indicated concentration range in a 384 - well format. Cells were then incubated with mIL - 3 agonist in 0.5% DMSO for 5 hrs 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 emission ratios were plotted against the indicated concentrations of Jak Inhibitor 1. Academic and non-profit customers, please inquire for special pricing.

BacMam Histone H3 [AcLys9] Cellular Assay Kit

The combination of baculovirus-mediated gene delivery (BacMam) with LanthaScreen® Cellular Assay technology enables a platform for the analysis of specific posttranslational modifications of histones. BacMam provides a convenient genetic delivery tool for a GFP-Histone H3 fusion protein in the cell line of interest. This kit describes an HTS-compatible cellular immunoassay measuring acetylation of GFP-Histone H3 at Lys9.

CellSensor™ HRE-bla ME-180 Cell Line

The CellSensor® HRE-bla ME-180 cell line contains a beta-lactamase reporter gene under control of the hypoxia response element (HRE) stably integrated into ME-180 cells. To construct this cell line, the HRE-bla construct was transduced into ME-180 cells by lentivirus. Flow cytometry was used to isolate cells responsive to deferoxamine. This cell line is validated for DMSO tolerance, cell number, stimulation time, substrate loading time, as well as Z' and EC50 concentrations for cobalt chloride and deferoxamine. The CellSensor® HRE-bla ME-180 cell line is responsive to deferoxamine and cobalt chloride and can be used to probe the hypoxia signaling pathway. This cell line can be adapted for high-throughput screening of agonist or antagonist 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™ Myc-bla HCT116 Cell Line

The CellSensor™Myc-bla HCT116 cell line contains a beta-lactamase reporter gene under the control of Myc binding sequences. The construct was transduced into HCT116 cells using a lentiviral system. HCT116 is a colon cancer cell line which expresses a mutated form of beta-catenin. This form of beta-catenin leads to the accumulation of beta-catenin and constitutive activation of downstream genes such as Myc. This cell line is a clonal population isolated by flow cytometry. It has been validated for cell plating density and DMSO tolerance. The signaling pathway has been validated using RNAi against c-Myc and ICG-001, an inhibitor of the wnt-beta-catenin pathway. The expression of the mutant beta-catenin in HCT116 cells results in constitutive activation of beta-lactamase in this CellSensor™line, which can be knocked down by ICG-001 (Figure 1) or Myc RNAi (Figure 2).

CellSensor™ c-Fos-bla HEK293T Cell Line

The c-fos-bla HEK 293T cell line contains a beta-lactamase reporter gene under control of the c-fos promoter stably integrated into HEK 293T cells. This cell line can be used to detect agonists/antagonists of the c-fos pathway. The c-fos-bla HEK 293T cells have been shown to be responsive to phorbol 12-myristate 13-acetate (PMA) stimulation. 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.

BacMam Histone H3K4me3 Cellular Assay Kit (Invitrogen™)

The BacMam Histone H3K4me3 Cellular Assay is a high-throughput screening (HTS) compatible TR-FRET assay for the interrogation of trimethylation at lysine 4 on histone H3 in a cellular format. Baculovirus-mediated gene delivery (BacMam) provides a convenient tool for the expression of GFP-Histone H3 fusion protein in your cell background of interest. Coupled with a terbium (Tb)-labeled site-specific antibody, the resulting cellular assay can be used to identify inhibitors of methyltransferases and demethylases acting on lysine 4 of histone H3.

The kit includes:

• BacMam Histone H3 Reagent
• LanthaScreen® Tb-anti-histone H3K4me3 Antibody
• 6X LanthaScreen® Cellular Lysis Buffer
• Instrument Control Terbium TR-FRET kit

The BacMam Histone H3K4me3 Cellular Assay Kit Lets You:
• Use your cell background of choice with the portability of BacMam
• Identify more relevant inhibitors since the methyl transferases and demethylases are in their natural protein complexes
• Conserve precious cell samples with the miniaturizable homogenous assay format
• Improve data quality with the advantages of TR-FRET

Get More Physiologically Relevant Results
The BacMam Histone H3K4me3 Cellular Assay allows the investigation of trimethylation at lysine 4 on histone H3 with your choice of cellular backgrounds including primary cells. This enables screening for potential inhibitors of methyl transferases and demethylases associated with Lys4 trimethylation in their natural complexes in a physiologically relevant cell type. Please note that the LanthaScreen® Tb-anti-Histone H3K4me3 antibody has been shown to have higher affinity for trimethylation compared to dimethylation at lysine 4 using a histone H3 peptide; however, when interpreting your results, it is important to consider that the antibody does show some affinity for the dimethylated peptide. Further discrimination between di- and trimethylation can be accomplished by using our complimentary BacMam Histone H3K4me2 Cellular Assay Kit (Cat # A14164), which is specific for dimethylation.

BacMam Convenience Saves Samples and Time
Assays are run in a fully homogenous, addition-only format without any of the washing, lysate transfer, or separation procedures required for traditional methods such as Western blotting and ELISA, making them ideal for HTS applications. By using the same BacMam reagent for GFP-Histone H3, other histone H3 modifications (Ph, Me, Ac) can be monitored just be exchanging the Terbium (Tb) labeled antibody. In addition, the application of the LanthaScreen® technology includes all the advantages of TR-FRET detection including reduced data noise, less interference from fluorescent compounds, and high sensitivity, allowing the use of fewer cells than traditional Western or ELISA methods.

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

CellSensor™ TNFa-bla THP-1 Cell Line

Tumor necrosis factor-alpha (TNFα) cytokine cell signaling is a critical pathway involved in immune and inflammatory responses. In addition, TNFα has been shown to inhibit growth and induce differentiation in tumor and non-tumor cells. The CellSensor™ TNFα-bla THP-1 cell line is a suspension cell line derived from human acute monocytic leukemia cells. The TNFα-bla THP-1 cell line is used to measure the TNFα cytokine signaling pathway via transcriptional regulation of the beta-lactamase gene. This cell line was constructed using GenomeScreen™ technology utilizing a promoterless beta-lactamase reporter gene randomly integrated into the genome of the THP-1 cell line. Sequential rounds of sorting were performed to isolate cell clones containing tagged genes induced by TNFα. These cells have been shown to be responsive to TNFa agonist. Academic and non-profit customers, please inquire for special pricing.

BacMam Akt [pSer473] Cellular Assay Kit

The combination of baculovirus-mediated gene delivery (BacMam) with LanthaScreen® Cellular Assay technology enables a platform for the analysis of specific posttranslational modifications of various substrates including AKT. BacMam provides a convenient genetic delivery tool for a GFP-AKT fusion protein in the cell line of interest. This kit describes an HTS-compatible cellular immunoassay measuring phosphorylation of GFP-AKT at Ser473.

CellSensor™ NFAT-bla CHO-K1 Cell Line

The G protein-coupled receptor (GPCR) superfamily is comprised of an estimated 600-1,000 members and is the largest known class of molecular targets with proven therapeutic value. GPCRs are instrumental in the transmission of a wide range of chemical messages from the extracellular environment to the interior of the cell. GPCRs are involved in a wide range of disorders, including allergies, cardiovascular dysfunction, depression, obesity, cancer, pain, diabetes, and various central nervous system disorders. GPCR signaling is mediated by trimeric G-proteins containing α, β, and γ subunits and can be categorized into signaling classes based upon subunit composition. G alpha, G alpha s, and G alpha i/o proteins mediate intracellular signaling through activation of signaling pathways leading to distinct physiological endpoints. Activation of G alpha s and G alpha i/o coupled receptors leads to stimulation or inhibition of adenylate cyclase, respectively; activation of G alpha q coupled receptors results in stimulation of phospholipase C. GPCR signaling through these distinct pathways can be monitored by activation of specific transcriptional response elements placed upstream of a reporter gene. Invitrogen has developed GeneBLAzer® Master Cell Lines employing the beta-lactamase reporter system, which can be used to screen for both agonists and antagonists of GPCRs coupling to each of the above signaling pathways. These cell-based assays make use of a membrane permeant fluorescent substrate (CCF4-AM or CCF2-AM), enabling a ratiometric readout that reduces errors due to sample variability, leads to excellent Z'-factor values, and allows for assay miniaturization. GeneBLAzer® Master Cell Lines with either NFAT or CRE response elements are available in Jurkat, CHO-K1, or FreeStyle™ 293F cell backgrounds (six cell lines in total) for the development of assays in suspension or adherent cell format. Stable cell lines employing an NFAT response element upstream of the beta-lactamase gene are designed for the study of Gq coupled receptors. Stable cell lines employing the cAMP response element (CRE) upstream of the beta-lactamase gene are designed for the study of Gs coupled receptors. FreeStyle™ 293F cells are HEK 293 cells that have been adapted for growth in suspension in serum-free media. Alternatively, these cells may be grown in the presence of serum for use in the adherent format. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ IL1-bla ECV304 Cell Line

The CellSensor® IL-1-bla ECV304 cell line is an adherent human urinary bladder cell line used to measure the (IL-1) signaling pathway via transcriptional regulation of the beta-lactamase gene. IL-1 cytokine cell signaling is a critical pathway involved in immune and inflammatory responses. This cell line was constructed using GenomeScreen™ technology utilizing a promoterless beta-lactamase reporter gene randomly integrated into the genome of the IL-1-bla ECV304 cell line. Sequential rounds of sorting were performed to isolate cell clones containing tagged genes induced by IL-1. A dose response assay was run with this cell line using IL-1 as the agonist. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ T-REx™ FOXO3 DBE-bla HeLa Cell Line

The CellSensor® T-REx™ FOXO3 DBE-bla HeLa cell line was engineered by lentiviral transduction of HeLa cervical cancer cells with a FOXO3 response element driving beta-lactamase expression (DBE-bla), along with a tetracycline repressor and tetracycline (or the tetracycline analog doxycycline)-inducible FOXO3 constructs. Addition of doxycycline to these cells allows for FOXO3 transcription factor expression and subsequent beta-lactamase expression, which in turn can be down-regulated by insulin-mediated activation of the PI3K/AKT signaling pathway which leads to phosphorylation and inactivation of FOXO3 and concomitant suppression of beta-lactamase expression. This cell line has been tested for robust assay performance by assessing a variety of assay parameters, including: cell plating number, DMSO tolerance, stimulation time, and substrate loading time. Assay validation was performed using serial dilutions of insulin to antagonize doxycycline- induced FOXO3 activity (Figure 1). Additional testing data using RNAi and alternate stimuli are also available.

CellSensor™ AP-1-bla A375 Cell Line

The CellSensor® AP1-bla A375 cell line contains a beta-lactamase reporter gene under control of the AP1 response element stably integrated into A375 cells. A375 cells are human melanoma cancer cells that contain the endogenous B-Raf mutation V600E resulting in constitutive B-Raf kinase activity. The CellSensor® AP1-bla A375 cell line is a clonal population isolated by flow cytometry based on constitutive expression of beta-lactamase. This cell line has been validated with various small-molecule Raf inhibitors as well as B-Raf Stealth RNAi™ siRNA. 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 Zfi-factor and IC50 concentrations of Raf1 Inhibitor I. The RAF gene family (RAF1, A-RAF, and B-RAF) encodes closely related serine/threonine protein kinases that are important effectors of Ras activation. Raf1 and A-Raf are rarely mutated, whereas mutations in B-Raf are common in human cancers, especially melanoma. B-Raf is mutated in about 70% of human melanomas, 35-70% of papillary thyroid carcinomas, and less commonly in lung and colorectal carcinomas. Mutations are mostly in the B-Raf kinase domain and, in melanomas, the vast majority are V600E missense mutations leading to activation of B-Raf kinase. The constitutive activity of B-Raf V600E can directly lead to the activation of the Mek/MAPK signaling pathway. Therefore, inhibition of B-Raf/Mek/MAPK signaling could be a potential way of treating melanomas and other tumors with mutant B-Raf.

CellSensor™ ISRE-bla Jurkat Cell Line

The CellSensor® ISRE-bla Jurkat cell line contains a beta-lactamase reporter gene under control of the interferon-stimulated response element (ISRE) stably integrated into Jurkat cells. To construct this cell line, the ISRE-bla construct was transduced into Jurkat cells by lentivirus. Flow cytometry was used to isolate cells responsive to IFNα. This cell line was validated for DMSO tolerance, cell number, stimulation time, substrate loading time, as well as ' and EC50 concentrations of IFN&alpha. The CellSensor® ISRE-bla Jurkat cell line is responsive to Interferon alpha (IFN-alpha) and Interferon beta and can be used to probe the type I interferon-induced JAK-STAT signaling pathways. This cell line can be adapted for high-throughput screening of agonist or antagonist compound libraries. Candidate drugs can also be tested for dose response against this cell line. ISRE-bla Jurkat cells were treated with agonist IFN-alpha; over the indicated concentration range in a 384-well format. Cells were incubated for 5 hours with agonist and 0.5% DMSO and then combined with LiveBLAzer™-FRET B/G Substrate (CCF4-AM) for 2 hours. Fluorescence emission values at 460 nm and 530 nm were obtained using a standard fluorescence plate reader, and the 460/530 ratios were plotted against the concentration of the agonist. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ C/EBP-bla FreeStyle™ 293F Cell Line

CCAAT⁄enhancer-binding protein (C⁄EBPa basic leucine zipper transcription factor, is involved in the regulation of mitotic growth arrest and differentiation. Mutations in C⁄EBPare thought to result in diseases such as human acute myeloid leukemia. C⁄EBPis expressed in liver, fat, lung, peripheral leukocytes, epidermis, intestine, and skeletal muscle. C⁄EBPalso plays a role in energy homeostasis, whereas dominant negative mutations cause altered hepatic glucose and glycogen metabolism as well as defects in white adipose tissue differentiation. The CellSensor® C⁄EBP-bla Freestyle™ 293F cell line contains a beta-lactamase reporter gene under control of the C⁄EBP response element stably integrated into Freestyle™ 293F cells. To construct this cell line, the C⁄EBP-bla construct was transduced into Freestyle™ 293F cells by lentivirus. Flow cytometry was used to isolate cells responsive to Phorbol 12-Myristate 13-Acetate (PMA). This cell line was tested under various conditions, including DMSO concentration, cell number, stimulation time, substrate loading time, and functionally validated for Z’ and EC50 concentrations using PMA. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ NFkB-bla RAW 264.7 Cell Line

The CellSensor® NFκB-bla RAW264.7 mouse macrophage cell line contains a beta-lactamase reporter gene under control of a stably integrated NFκB response element. This cell line is a clonal population isolated in response to lipopolysaccharide by flow cytometry. This cell line has been tested for assay performance under variable conditions, including DMSO concentration, cell number, stimulation time, and substrate loading time, and has been validated for Zfi-factor and EC50 concentrations of the TLR7 ligand imiquimod. Additional data using alternate stimuli are shown. The molecules responsible for coordinating the immune systems recognition of pathogens are Toll-like receptors (TLRs), of which there are currently 10 identified members in humans. TLRs are pattern recognition receptors (PRRs), which bind to pathogen-associated molecular patterns (PAMPs), small molecular sequences consistently found on pathogens. This binding activates the transcription of immune system genes and regulators such as cytokines, chemokines, and costimulatory molecules, thereby promoting the initial immune response of macrophages and neutrophils. Academic and non-profit customers, please inquire for special pricing.

GeneBLAzer™ Jurkat Control Kit

Th e GeneBLAzer® Jurkat Control Kit contains constitutively expressing (CMV-bla) and wildtype Jurkat cells to provide positive and negative controls, respectively, for evaluating beta-lactamase activity (Figure 1).

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 Akt [pThr308] Cellular Assay Kit

The combination of baculovirus-mediated gene delivery (BacMam) with LanthaScreen® Cellular Assay technology enables a platform for the analysis of specific posttranslational modifications of various substrates including AKT. BacMam provides a convenient genetic delivery tool for a GFP-AKT fusion protein in the cell line of interest. This kit describes an HTS-compatible cellular immunoassay measuring phosphorylation of GFP-AKT at Thr308.

LanthaScreen™ PDCD4 HEK 293E Cell Line

The PI3K⁄AKT⁄mTOR pathway mediates signals for cell growth and survival, transcription and translation, and regulated cell death. The significance of mTOR has been underscored recently by the identification of two distinct cellular complexes: mTORC1 (includes raptor and is rapamycin sensitive) and mTORC2 (includes rictor and is insensitive to rapamycin). Activation of the pathway by insulin (or other mitogens) leads to the phosphorylation of programmed cell death protein 4 (PDCD4) via mTORC1 (indirectly through the ribosomal protein p70 S6 kinase). LanthaScreen® PDCD4 HEK293E is a human cell line which constitutively expresses GFP-PDCD4 fusion proteins. This kinase target was introduced using lipid transfection followed by selection with Blasticidin. These cells are a clonal population isolated by FACS using GFP fluorescence as a sorting marker. A homogenous immunoassay ("addition only", no wash steps) was developed in 384-well format using a TR FRET-based detection method to monitor the phosphorylation of Ser457 on GFP-PDCD4 using a terbium-labeled phosphospecific antibody. The assay has been optimized for performance under numerous experimental conditions (e.g., cell plating density, DMSO tolerance, insulin stimulation time, etc.) and displays excellent statistical data (Z' > 0.6). Moreover, this cell line has been validated with different stimuli and known inhibitors (shows correct EC50 and IC50 values) and serves as robust cellular readout for mTORC1 signaling.

CellSensor™ LEF/TCF-bla HCT-116 Cell Line

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 activating the Frizzled transmembrane receptors transduced the signal to a cytoplasmic protein, known as disheveled protein, which then inhibits the serine/threonine kinase Glycogen Synthase-3 Beta (GSK-3B). This signal leads to functional inactivation and dissociation of a multi-protein Beta -catenin destruction comples, which is made up of the tumor suppressor protein Adenomatous Polyposis Coli (APC), GSK-3B and a scaffold of Axin. This results in dephosphorylation and dissociation of Beta -catenin. The unphosphorylated b-catenin is stabilized and accumulates in the cytoplasm of the cell. Beta -catenin then associates with the T-Cell Factor (TCF)/Lymphoid Enhancer Factor (LEF) 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. The CellSensor™ LEF/TCF-bla HCT-116 cell line contains a beta-lactamase reporter gene under control of the LEF/TCF stably integrated into HCT-116 cells. HCT-116 is a colon cancer cell line carrying a gain-of-function mutation in beta-catenin gene (deletion of amino acid Serine45), which prevents beta-catenin protein degradation leading to constitutive activation of downstream genes. Thus, this cell line constitutively expresses beta-lactamase, which may be further stimulated or inhibited, e.g., using Stealth RNAi™ siRNA. This cell line has also been tested under various experimental conditions, including DMSO concentration, cell number, stimulation time, and substrate loading time. Academic and non-profit customers, please inquire for special pricing.

BacMam Aequorin Kit (Invitrogen™)

The BacMam Aequorin Kit utilizes a modified baculovirus (insect cell virus with a mammalian promoter) to transiently deliver aequorin, a luminescent calcium sensor, into mammalian cells from a wide variety of sources: cell lines, primary cells, and stem cells. Upon binding calcium ions, aequorin displays a "flash" style luminescent signal while consuming coelenterazine as a substrate. The luminescence signal resolves problems associated with autofluorescent compounds in traditional assays and provides an exceptional signal-to-noise ratio.

The BacMam Aequorin Kit Allows You to:

Measure calcium in a wide variety cell types

• Develop an assay in 2-3 weeks rather than months
• Take advantage of a large assay window
• Avoid false positives due to autofluorescent compounds

Study Signaling in the Most Relevant Cellular Background
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 Aequorin 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.

Save Time and Money with Faster Assay Development
The BacMam Aequorin Kit 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 Aequorin a user can get results in 2-3 weeks versus months.

Design the Assay to Meet Your Requirements
The BacMam Aequorin Kit provides flexibility to your research, allowing you to understand signaling across various cell backgrounds. BacMam Aequorin can interrogate calcium signaling in both GPCR and Ion Channel targets whether endogenously or exogenously introduced. To gain even greater flexibility, transduce large quantities of cells in batch mode and store frozen transduced cells in aliquots to use at your convenience.

Obtain High Quality Results with a Large Dynamic Range
BacMam Aequorin is a luminescent calcium indicator. Luminescence readouts present little to no background signal, increasing the robustness of the assay; and they have a large assay window. The assay uses luminescence, thereby minimizing the number of false positive hits due to compound autofluorescence.

BacMam Aequorin Kit Compatible Cells
The BacMam Aequorin Kit has been used to successfully transduce numerous transformed cell lines (e.g. U2-OS, HEK 293, CHO-K1 and HeLa), primary cells (e.g., fibroblasts, HASMC and HUVEC), 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.

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

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.

CellSensor™ CRE-bla HEK 293T Cell Line

The CellSensor® CRE-bla HEK 293T cell line contains the b-lactamase reporter gene under control of the cAMP response element (CRE). To obtain the cell line, the CRE-bla construct was transduced into HEK 293T cells by lentivirus. Subsequent flow cytometry was used to isolate clones responsive to forskolin stimulation. The CellSensor® CRE-bla HEK 293T cell line can be used to build specific G-protein coupled receptor (GPCR) assays or to detect changes in intracellular cAMP levels. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ STAT6-bla RA-1 Cell Line

The CellSensor® STAT6-bla RA-1 Cell Line contains a beta-lactamase reporter gene under control of the STAT6 response element stably integrated into Ramos-1 (RA-1) cells. This cell line is a clonal population isolated by flow cytometry in response to interleukin-4 (IL-4). This cell line has been tested for assay performance under variable conditions, including DMSO concentration, cell number, stimulation time, and substrate loading time, and validated for Zfi-factor and EC50 concentrations of IL-4. Additional data using alternate stimuli are also available. IL-4 is known to induce the growth and differentiation of B cells, T cells, myeloid cells, and hepatocytes. In B cells, IL-4 acts as a co-mitogen to induce the expression of the Fc receptor for IgE and also major histocompatibility complex (MHC) class II molecules, and it stimulates the transcription of immunoglobulin heavy-chain germline IgE and IgG1 genes, leading to class switching. IL-4 function is mediated by the IL-4 receptor complex, which activates Jak1 and Jak3 tyrosine kinases. STAT6 is recruited to the IL-4R complex and is subsequently phosphorylated by the Jak kinases. This causes STAT6 to dimerize and translocate to the nucleus where it binds to specific sequences on IL-4-responsive genes. Optimal transcription response of IL-4-inducible promoters requires two Th2-mediated stimuli, CD40 ligand and IL-4. CD40 ligand is a protein expressed on the surface of T cells. It regulates B cell function by engaging CD40 on the B cell surface, leading to signaling through NFκB. Academic and non-profit customers, please inquire for special pricing.

LanthaScreen™ STAT1 U2OS Cell Line

The JAK/STAT1 signaling pathway is known to be activated by type I/II interferons such as interferon-gamma and interferon-alpha. In this pathway, binding of these cytokines to their respective cell-surface receptors results in the activation of JAKs, which in turn phosphoactivate STAT1 proteins at a specific tyrosine residue (Y701). LanthaScreen® STAT1 U2OS is a human cell line that constitutively expresses a GFPSTAT1 fusion protein. The JAK/STAT1 signaling pathway is known to be functionally intact in this cell line; therefore, the GFP-STAT1 fusion protein serves as a substrate for the IFN-gamma-inducible phosphorylation by JAKs. Using this cell line, a homogeneous immunoassay has been developed in which the phosphorylation state of GFP-STAT1 is detected in cell lysates using a terbium-labeled anti-pY701-STAT1 antibody in a time-resolved FRET (TR-FRET) readout. The GFP-STAT1 DNA expression construct was transfected into U2OS cells using Lipofectamine™ LTX Reagent, followed by selection with blasticidin. This cell line is a clonal population isolated by flow cytometry using GFP fluorescence as a sorting marker. Using the lytic TR-FRET immunoassay, this cell line is validated for EC50 and Zfi-factors under optimized conditions using IFN-gamma as a ligand for JAK-mediated GFP-STAT1 phosphorylation. This assay has also been tested for assay performance under variable experimental conditions, including cell plating density, stimulation time, DMSO tolerance, and assay development time. Additional information using alternate stimuli and alternate assay protocols is available.

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™ DHFR/E2F-bla NIH3T3 Cell Line

The CellSensor® dhfr(E2F)-bla NIH 3T3 Cell Line contains a beta-lactamase reporter gene under control of the E2F/DP1 binding sequence from the DHFR gene promoter, stably integrated into NIH 3T3 cells. This cell line is a clonal population isolated by flow cytometry in response to 10% newborn bovine serum. This cell line has been tested for assay performance under variable conditions, including DMSO concentration, cell number, stimulation time, and substrate loading time, and validated for Z-factor using newborn bovine serum. Additional data using alternate stimuli are shown. The G1/S cell-cycle checkpoint controls the passage of eukaryotic cells from the first gap phase (G1) into the DNA synthesis phase (S). Two cell-cycle kinases, CDK4/6-cyclin D and CDK2-cyclin E, and the transcription complex that includes Rb and E2F, are pivotal in controlling this checkpoint. During G1, the Rb-HDAC repressor complex binds to the E2F-DP1 transcription factors, inhibiting downstream transcription. Phosphorylation of Rb by CDK4/6 and CDK2 dissociates the Rb-repressor complex, permitting transcription of S-phase genes encoding for proteins that amplify the G1-to-S switch and that are required for DNA replication. Many different stimuli exert checkpoint control, including TGFβ, DNA damage, contact inhibition, replicative senescence, and growth factor withdrawal. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ irf1-bla TF-1 Cell Line

Jak/Stat signaling pathways play essential roles in 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 TF1 Cell Line contains a beta - lactamase reporter gene under control of the irf - 1 response element stably integrated into TF1 cells. TF1 cells are a human erythroleukemia cell line that is growth - dependent on GM - CSF and have an intact GM - CSF - JAK2 - STAT5 pathway. This cell line is validated for EC50 and Z’ - factor using GM - CSF. This cell line has also been tested under variable experimental conditions, including DMSO concentration, cell number, stimulation time, and substrate loading time. Responsiveness to EPO and Jak Inhibitor 1 was also tested (Figures 2 and 3). CellSensor® irf1 - bla TF1 cells were stimulated in triplicate with GM - CSF over the indicated concentration range in a 384 - well format. Cells were incubated for 5 hrs with agonist and 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 were plotted for each replicate against the indicated concentrations of GM - CSF. CellSensor® irf1 - bla TF1 cells were stimulated with Epo over the indicated concentration range in a 384 - well format. Cells were incubated for 5 hrs with agonist and 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 Response Ratios were plotted against the indicated concentrations of Epo. CellSensor® irf1 - bla TF1 cells were treated with Jak Inhibitor 1 over the indicated concentration range in a 384 - well format. Cells were then stimulated with GM - CSF or EPO for 5 hrs 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. These values were converted to percent inhibition relative to a set of controls (unstimulated cells and EC80 of GM - CSF or EPO treated cells) and plotted against the indicated concentrations of Jak Inhibitor 1. Academic and non-profit customers, please inquire for special pricing.

LanthaScreen™ PRAS40 293E Cell Line

The PI3K/AKT pathway is activated by various stimuli (including insulin and IGF-1) and mediates signals for cell growth, cell survival, translation, and inhibition of apoptosis. PRAS40 (Proline-rich AKT substrate, 40 kDa) is a cytosolic protein that is phosphorylated at position Thr246 by AKT directly upon stimulation of the pathway. PRAS40 has been shown to bind to the mTORC1 complex and inhibit downstream signaling (i.e., phosphorylation of 4E-BP1 or p70S6K). LanthaScreen® PRAS40 HEK293E is a human cell line which constitutively expresses GFP-PRAS40 fusion proteins. The kinase substrate
was introduced using lentiviral transduction followed by selection with blasticidin. This cell line is a clonal population isolated by flow cytometry using GFP fluorescence as a sorting marker. The PI3K/AKT pathway is known to be active in the highly insulin-responsive HEK293E cell background. Using this cell line, a homogenous ("addition only") immunoassay has been developed in a 384-well format. The phosphorylation of PRAS40 at Thr246 is detected in cell lysates using a terbium-labeled phosphospecific antibody in a time-resolved FRET (TR-FRET) readout. The performance of this assay has been tested using numerous experimental variables, including cell plating density, stimulation time, DMSO tolerance, and antibody incubation time. Under optimized conditions, the assay has been further validated and shows correct EC50 and IC50 values, with insulin as the primary agonist and PI-103 as the known inhibitor. Overall, the assay displays excellent statistical data (Z'-factor >0.6), high signal-to-background (response ratio), and is a robust cell-based readout of AKT signaling.

CellSensor™ CRE-bla Jurkat Cell Line

The CRE-bla Jurkat cell line contains a beta-lactamase reporter gene under control of a cAMP response element (CRE) stably integrated into Jurkat 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 Jurkat cells have been shown to be responsive to forskolin stimulation. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ ISRE-bla RA-1 Cell Line

The CellSensor® ISRE-bla RA-1 Cell Line contains the beta-lactamase (bla) gene under the control of the interferon-stimulated response element (ISRE). This cell line was engineered by lentiviral transduction of an ISRE-bla construct into RA-1 cells. Flow cytometry was then used to isolate a pool of cells responsive to polyinosinic-polycytidylic acid (poly I:C) stimulation. ISRE-bla RA-1 cells have been tested for assay performance using variable assay conditions, including DMSO concentration, cell number, stimulation time, substrate loading time, as well as validated for Z'-factor and EC50 concentrations of poly I:C. Additional testing data using alternate stimuli are also available. Academic and non-profit customers, please inquire for special pricing.

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.

CellSensor™ LEF/TCF-bla FreeStyle™ 293F Cell Line

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. Binding of the Wnt protein to the Frizzled transmembrane receptors initiates a signaling cascade that activates the Dishevelled protein, which then inhibits the serine/threonine kinase Glycogen Synthase - 3 Beta (GSK - 3B). This signal leads to functional inactivation and dissociation of a multiprotein beta - catenin destruction complex made up of the tumor suppressor protein Adenomatous Polyposis Coli (APC), GSK - 3B, 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 Lymphoid Enhancer Factor (LEF)/T - Cell Factor (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. The CellSensor® LEF/TCF - bla FreeStyle™ 293 Cell Line contains a beta - lactamase reporter gene under control of the LEF/TCF stably integrated into FreeStyle™ 293 cells. This cell line is validated for EC50 and Z' - factor under optimized conditions using mWnt3a. This cell line has also been tested under various experimental conditions, including DMSO concentration, cell number, stimulation time, and substrate loading time. CellSensor® LEF/TCF - bla FreeStyle™ 293F cells were stimulated with mWnt3a over the indicated concentration range in a 384 - well format. Cells were incubated for 5 hrs with agonist and 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 are shown plotted against the indicated concentrations of mWnt3a. Academic and non-profit customers, please inquire for special pricing.

GeneBLAzer™ CMV-bla Jurkat Positive Control Cell Line

The GeneBLAzer® CMV-bla Jurkat Cell Line constitutively expresses beta-lactamase under the control of the CMV promoter in Jurkat cells. A stable population was obtained using 1 mg/ml Geneticin® (G418) selection followed by isolation of a clone by fluorescence-activated cell sorting (FACS).

CMV-bla Jurkat cells can be used as a positive control for beta-lactamase expression experiments. When the cells are loaded with CCF2-AM or CCF4-AM substrate and excited with 409 nm light, the cells will fluoresce blue.

LanthaScreen™ Akt HEK 293E Cell Line

LanthaScreen® GFP Cellular Assays allow for the analysis of post-translational modifications for a number of target proteins in an live-cell context. The PI3K⁄AKT pathway mediates signals for cell growth, cell survival, transcription, translation, and glucose uptake. Because of the complexity of this signaling cascade, especially as applied to the regulation of the mammalian target of rapamycin (mTOR), cell-based methods are critical for proper identification of small-molecule mediators of this pathway. The significance of mTOR as a kinase has been underscored recently by the identification of two distinct multimeric complexes inside the cell: mTORC1 (includes raptor and is rapamycin sensitive) and mTORC2 (includes rictor and is insensitive to acute rapamycin exposure). mTORC2 has been shown to phosphorylate and AKT at residue Ser473 for complete activation of this prosurvival kinase. LanthaScreenTM AKT HEK293E is a human cell line which constitutively expresses GFP-AKT fusion proteins. This kinase target was introduced using lipid transfection and these cells are a clonal population isolated by FACS, using GFP 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 Ser473 on GFP-AKT is detected in cell lysates using a terbium-labeled phosphor-specific antibody. 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.

CellSensor™ irf1-bla HEL Cell Line

Jak/Stat signaling pathways play essential roles in the cellular responses to distinct cytokines. One of 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 knock-out causes embryonic lethality due to defective erythropoiesis, suggesting the Jak2/Stat5 pathway plays important role in red blood cell formation. Recent discovery of activating mutation in JAK2 (V617F) present in high percentage of myeloproliferative disease (MPD) patients suggests Jak2/Stat5 pathway to be the potential therapeutic target for certain forms of MPD. The activated transcription factor Stat5 dimers recognize and bind 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 HEL cell line contains a beta-lactamase reporter gene under control of the interferon regulatory factor-1 (irf1) response element stably integrated into HEL cells. HEL cells are a human erythroleukemia cell line that is growth factor independent and contains a endogenous homozygous JAK2V617F mutation. This cell line validated for IC50 and Z'-Factor under optimized conditions using Jak Inhibitor 1 (Figure 1). This cell line has also been tested under variable experimental conditions, including DMSO concentration, cell number, stimulation time, and substrate loading time. Academic and non-profit customers, please inquire for special pricing.

LanthaScreen™ ATF2 (19-106) A549 Cell Line

The LanthaScreen®ATF2 (19-106) A549 cell line contains a fusion protein consisting of GFP and a fragment encoding for aa 19-106 of ATF2 under the control of a CMV promotor. A549 is a lung carcinoma cell line which shows inducible activation of JNK by number of different ligands, such as TNF and EGF, which leads to the transient phosphorylation of GFP-ATF2 (19-106). This LanthaScreen®cell line therefore allows the analysis of JNK activity using GFP-ATF2 phosphorylation as readout. The GFP-ATF2 (19-106) construct was transfected into A549 cells using Lipofectamine™2000, followed by selection with Blasticidin. This cell line is a clonal population isolated by flow cytometry using GFP fluorescence as sorting marker. This cell line is validated for EC50 and Z' under optimized conditions using TNF as ligand for JNK mediated GFP-ATF2 (19-106) phosphorylation. This cell line has been tested for assay performance under variable experimental conditions, including cell plating density, stimulation time, DMSO tolerance and assay development time. Additional information using alternate stimuli is also available. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ T-REx™ NICD CSL-bla HeLa Cell Line

The CellSensor® T-REx™ NICD CSL-bla HeLa cell line was engineered by lentiviral transduction of HeLa cervical cancer cells with a notchresponse element driving beta-lactamase reporter gene expression (CSL-bla) along with tetracycline repressor and tetracycline (or the tetracycline analog, doxycycline)-inducible NICD (notch intracellular domain) constructs. This cell line is a clonal population isolated by flow cytometry. Addition of doxycycline to these cells allows for regulated NICD transcription factor expression and subsequent beta-lactamase expression. This cell line has been tested for robust assay performance by assessing a variety of parameters, including cell plating number, DMSO tolerance, stimulation time, and substrate loading time. Assay validation was performed using serial dilutions of doxycycline. Additional testing data using RNAi and alternate stimuli are also available.

CellSensor™ CRE HEK 293T DA Assay Kit

The CellSensor® CRE HEK 293T DA (Division Arrested) cells contains a beta-lactamase (bla) reporter gene under control of a cAMP response element (CRE) stably integrated into HEK293T cells. Fluorescence activated cell sorting (FACS) was used to isolate clones responsive to stimulation of the CRE pathway by forskolin. The cell line was validated for DMSO tolerance, cell number, stimulation time, substrate loading conditions, and EC50 concentration of forskolin. DA cells are irreversibly division arrested using a low-dose treatment of Mitomycin C, and have no apparent toxicity or change in cellular signal transduction.

This product can serve as a negative control in screening assays performed with specific G-protein coupled receptor (GPCR) DA cells that were made from this same background. These cells can be used in other assays where a sensitive readout to intracellular changes to cAMP is needed.

CellSensor™ NFκB-bla THP-1 Cell Line

The CellSensor® NFκB-bla THP-1 cell line contains a beta-lactamase reporter gene under the control of the Nuclear Factor kappa B (NFκB) response element stably integrated into THP-1 cells. To obtain the cell line, the NFκB-bla construct was transduced into THP-1 cells by lentivirus. Subsequent flow cytometry was used to isolate cells responsive to Tumor Necrosis Factor alpha (TNFα). The cell line was validated for DMSO tolerance, cell number, stimulation time, substrate loading time, and EC50 concentration of TNF&alpha. The CellSensor® NFκB-bla THP-1 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™ c-Fos-bla ME-180 Cell Line

The CellSensor® c-fos-bla ME-180 cell line contains a beta-lactamase reporter gene under control of the c-fos response element stably integrated into ME-180 cells. To construct this cell line, the c-fos-bla construct was transduced into ME-180 cells by lentivirus. Flow cytometry was used to isolate cells responsive to epidermal growth factor (EGF). This cell line is validated for DMSO tolerance, cell number, stimulation time, substrate loading time, as well as Z' and EC50 concentrations for EGF. The CellSensor® c-fos-bla ME-180 cell line is responsive to EGF, HGF, IL-6, OSM, TNFα, IL-1-alpha; and PMA/Thaps, and can be used to probe the JNK/P38/MAPK and JAK/STAT signaling pathways. This cell line can be adapted for high-throughput screening of agonist or antagonist 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™ p53RE-bla HCT-116 Cell Line

The CellSensor® p53RE-bla HCT-116 cell line contains a beta-lactamase reporter gene under control of the p53 response element stably integrated into HCT-116 cells. To construct this cell line, the p53RE-bla construct was transduced into HCT-116 cells by lentivirus. Flow cytometry was used to isolate cells responsive to Mitomycin. This cell line was validated for DMSO tolerance, cell number, stimulation time, substrate loading time, as well as Z'; and EC50 concentrations for Mitomycin. The CellSensor® p53RE-bla HCT-116 cell line is responsive to Mitomycin and can be used to probe p53 signaling, which is involved in DNA repair, cell cycle arrest, and in some cases, apoptosis. 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™ TrkC-NFAT-bla CHO-K1 Cell Line

Neurotrophins (NGF, BDNF, NT-3, and NT-4) and their transmembrane receptors (TrkA, TrkB, TrkC, and P75NTR) play important roles in the regulation of neuronal and non-neuronal cell proliferation, differentiation, survival, and death. Neurotrophin signaling also mediates higher-order neuronal activities, such as learning, memory, and behavior. Alterations in neurotrophin levels and their receptors have been implicated in neurodegenerative disorders such as Alzheimer's disease and Huntington's disease, as well as psychiatric disorders. Neurotrophins propagate their signal through activating multiple signaling pathways. One of the signaling pathways of NT-3 (the ligand for TrkC) activates phospholipase C, releasing DAG and IP3, increasing downstream intracellular calcium, and activating protein kinase C. This in turn promotes the translocation of the transcription factor-nuclear factor of activated T-cells (NFAT)-from the cytosol into the nucleus, resulting in NFAT-dependent transcription. The CellSensor® TrkC-NFAT-bla CHO-K1 Cell Line was engineered by integrating the human TrkC expression plasmid into the genome of the CellSensor® NFAT-bla CHO-K1 cell line, which is engineered to express beta-lactamase under the control of NFAT. This cell line has been tested for assay performance under variable conditions, including DMSO concentration, cell number, stimulation time, substrate loading time, and in cryopreserved cells, and has been validated for Z'-factor and EC50 values under optimized conditions using NT-3. Additional testing information using various small-molecule inhibitors and RNAi has been performed.

CellSensor™ AP-1-bla HEK293T Cell Line

The CellSensor® AP-1-bla HEK 293T Cell Line contains a beta-lactamase reporter gene under control of the AP-1 response element stably integrated into HEK 293T cells. The cell line was created through sorting by FACS of cells responsive to stimulation of the AP-1 pathway with phorbol 12-myristate 13-acetate (PMA). The cell line was validated for DMSO tolerance, incubation time with stimulant, and substrate loading conditions. The AP-1-bla HEK 293T cell line responds to agonist treatment as expected from 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.

BacMam Histone H3K27me3 Cellular Assay Kit (Invitrogen™)

The BacMam Histone H3K27me3 Cellular Assay is a high-throughput screening (HTS) compatible TR-FRET assay for the interrogation of trimethylation at lysine 27 on histone H3 in a cellular format. Baculovirus-mediated gene delivery (BacMam) provides a convenient tool for the expression of GFP-Histone H3 fusion protein in your cell background of interest. Coupled with a terbium (Tb)-labeled site-specific antibody, the resulting cellular assay can be used to identify inhibitors of methyltransferases and demethylases acting on lysine 27 of histone H3.

The kit includes:

• BacMam Histone H3 Reagent
• LanthaScreen® Tb-anti-histone H3K27me3 Antibody
• 6X LanthaScreen® Cellular Lysis Buffer
• Instrument Control Terbium TR-FRET kit

With the BacMam Histone H3K27me3 Cellular Assay Kit You Can:
• Use your cell background of choice with the portability of BacMam
• Identify more relevant inhibitors since the methyl transferases and demethylases are in their natural protein complexes
• Conserve precious cell samples with the miniaturizable homogenous assay format
• Improve data quality with the advantages of TR-FRET

Get More Physiologically Relevant Results
The BacMam Histone H3K27me3 Cellular Assay allows the investigation of trimethylation at lysine 27 on histone H3 with your choice of cellular backgrounds including primary cells. This enables screening for potential inhibitors of methyl transferases and demethylases associated with Lys27 trimethylation in their natural complexes in a physiologically relevant cell type.

BacMam and LanthaScreen® Convenience Saves Sample and Time
Assays are run in a fully homogenous, addition-only format without any of the washing, lysate transfer, or separation procedures required for traditional methods such as Western blotting and ELISA, making them ideal for HTS applications. By using the same BacMam reagent for GFP-Histone H3, other histone H3 modifications (Ph, Me, Ac) can be monitored just be exchanging the Terbium (Tb) labeled antibody. In addition, the application of the LanthaScreen® technology includes all the advantages of TR-FRET detection including reduced data noise, less interference from fluorescent compounds, and high sensitivity, allowing the use of fewer cells than traditional Western or ELISA methods.

CellSensor™ SBE-bla HEK 293T Cell Line

Smad cell signaling is a critical pathway involved in cell growth and proliferation. The CellSensor® SBE-bla HEK 293T cell line is responsive to transforming growth factor-beta 1 (TGF-ß1) and can be used to probe the Smad signaling pathway.The CellSensor® SBE-bla HEK 293T cell line contains the !-lactamase reporter gene under the control of the Smad Binding Element (SBE). The SBE-bla construct was transduced into HEK 293T cells by lentivirus to obtain the cell line. Flow cytometry was used to isolate a clone responsive to TGF-ß1 a dose response curve for this cell line using TGF-B1 is available in the validation packet data. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ NFAT bla HEK 293T Cell Line

The CellSensor® NFAT-bla HEK 293T cell line contains a beta-lactamase reporter gene under the control of the Nuclear Factor of Activated T Cells (NFAT) response element stably integrated into HEK 293T cells. Fluorescence activated cell sorting (FACS) was used to isolate clones responsive to stimulation of the NFAT pathway by PMA (Phorbol 12-Myristate 13-Acetate) and Thapsagargin (Thaps). The cell line was validated for DMSO tolerance, cell number, stimulation time, substrate loading conditions, and EC50 concentration of Thaps at fixed concentration of PMA. The CellSensor® NFAT-bla HEK 293T cell line responds to agonist treatment as expected from the literature and can be adapted for high-throughput screening (including coupling of receptors to the NFAT pathway) for agonists or antagonists 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.

LanthaScreen™ IkB alpha GripTite™ Cell Line

The LanthaScreen®IkB alpha GripTite® cell line contains a fusion protein consisting of the cDNA encoding for GFP and IkB (GFP is fused to the N-terminus of IkB alpha) under the control of a CMV promoter stably transfected into GripTite® cells. GripTite® is a modified HEK293 cell line, which is a human kidney cell line which shows inducible activation of the NFkB signaling pathway by a number of different ligands, such as TNF and IL-1. Activation of the NFkB pathway involves the activation of IKK resulting in the phosphorylation of its target protein IkB alpha. This LanthaScreen cell line allows therefore the analysis of IKK activity using GFP-IkB alpha phosphorylation as readout. The GFP-IkB alpha construct was transfected into GripTite® cells using Lipofectamine™2000, followed by selection with Blasticidin. This cell line is a clonal population isolated by flow cytometry using GFP fluorescence as sorting marker. This cell line is validated for EC50 and Z' under optimized conditions using TNF as ligand for IKK mediated GFP-IkB alpha phosphorylation or ubiquitination. This cell line has also been tested for assay performance under variable experimental conditions, including cell plating density, stimulation time, DMSO tolerance and assay development time. Additional information using alternate stimuli is also available. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ CRE-bla FreeStyle™ 293F Cell Line

The CellSensor® CRE-bla FreeStyle™ 293F cell line contains a beta-lactamase reporter gene under control of the CRE response element stably integrated into FreeStyle™ 293F cells. To construct this cell line, the CRE -bla construct was transduced into FreeStyle™ 293F cells by lentivirus. Flow cytometry was used to isolate cells responsive to forskolin. Assay development experiments performed on this cell line included DMSO tolerance, cell number, stimulation time and substrate loading time. Functional validation of this cell line under optimized conditions included and Z'; and EC50 determinations using forskolin as a primary agonist. The CellSensor® CRE-bla FreeStyle™ 293F cell line is responsive to forskolin and can be used to build specific G-protein coupled receptor (GPCR) assays after transfection of additional genes of interest or to detect changes in cellular cAMP levels. 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™ ARE-bla Hep G2 Cell Line

The CellSensor® ARE-bla Hep G2 cell line is responsive to tBHQ and Sulforaphane and can be used for analyzing the Nrf2/antioxidant response signaling pathway using the GeneBLAzer® Loading Substrates. Reactive oxygen species (ROS) can damage biological macromolecules and are detrimental to cellular health. Electrophilic compounds, xenobiotics and antioxidants are sources of reactive oxygen species, creating oxidative stress that can harm cells. Enzymes are involved in the Phase II detoxification of xenobiotics to reduce cellular stress include glutathione transferases, quinone reductase, epoxide hydrolase, heme oxygenase, UDP-glucuronosyl transferases, and gamma-glutamylcysteine synthetase. Expression of these genes protects cells from oxidative damage and can prevent mutagenesis and cancer. Transcription of these enzymes is coordinately regulated through antioxidant response elements (AREs). Nrf2 (NF-E2-related factor 2) and Nrf1 are transcription factors that bind to AREs and activate these genes. The CellSensor™ ARE-bla Hep G2 cell line contains a beta-lactamase reporter gene under control of the Antioxidant Response Element (ARE) stably integrated into Hep G2 cells. To construct this cell line, the ARE-bla construct was transduced into Hep G2 cells. Flow cytometry was used to isolate cells responsive to tert-butylhydroquinone (tBHQ). This cell line is tested under various experimental conditions, including DMSO concentration, cell number, stimulation time, substrate loading time, and functionally validated for Z'; and EC50 concentrations of tBHQ. Academic and non-profit customers, please inquire for special pricing.

LanthaScreen™ STAT5 (JAK2 V617F) U2OS Cell Line

LanthaScreen® GFP Cellular Assays allow for the analysis of post-translational modifications for a number of target proteins in an live-cell context. The JAK2⁄STAT5 signaling pathway plays an essential role in blood cell formation in response to cytokines such as GM-CSF, IL-3, and EPO. The recent discovery of an activating mutation in JAK2 (V617F) present in high percentage of myeloproliferative disease (MPD) patients suggests that this mutant JAK2 activity is a potential therapeutic target for certain forms of MPD. The assay described in this summary makes use of a cell line engineered for expression of the constitutively-active mutant kinase JAK2 V617F. By co-expressing GFP-STAT5 in this background, the phosphorylation state of STAT5 (specifically residue Tyr 694⁄699) can be modulated with JAK2 V617F inhibitors and analyzed in cell lysates using an anti-STAT5 [pTyr 694⁄699] and LanthaScreenTM terbium-anti-mouse antibody pair. GFP-STAT5 Lentivirus was transduced into U2OS cells followed by selection with Blasticidin. The selected pool was then transfected with a GST-JAK2 V617F construct using LipofectamineTM LTX, followed by selection with Geneticin. This cell line is a clonal population isolated by flow cytometry using GFP fluorescence as sorting marker, and has been screened for the constitutive expression of GFP-STAT5 and GST-JAK2 V617F. Using a lytic TR-FRET immuno-assay, this cell line is validated for IC50 and Z' under optimized conditions using JAK Inhibitor I (Pyridone 6) as a small molecule inhibitor for JAK2 V617F-mediated GFP-STAT5 phosphorylation. This assay has also been tested for assay performance under variable experimental conditions, including cell plating density, stimulation time, DMSO tolerance and lysis⁄equilibration time.

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.