Cellsensor Cellular Pathway Assays

CellSensor™ TrkC-NFAT-bla CHO-K1 Cell Line Thermo Scientific™

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™ TrkA-NFAT-bla CHO-K1 Cell Line Thermo Scientific™

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™ Gli-bla 22Rv.1 Cell Line Thermo Scientific™

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™ CRE-bla CHO K1 Cell Line Thermo Scientific™

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™ irf1-bla TF-1 Cell Line Thermo Scientific™

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.

CellSensor™ HSE-bla HeLa Cell Line Thermo Scientific™

Activation of the heat shock response⁄unfolded protein response (HSR⁄UPR) occurs in response to a diversity of chemical, environmental, and physiological stress conditions. Transcriptional regulation of the human HSR is mediated by a family of three heat shock transcription factors (HSFs), HSF-1, -2, and -4. Stress-induced activation of quiescent HSF monomers results in their trimerization and accumulation in the nucleus, wherein they bind to and upregulate transcription of target genes (e.g. molecular chaperones, certain proteases, and other stress response genes) harboring a heat shock element (HSE). Downstream expression of heat shock protein family members (e.g. Hsp90 and Hsp70) that function as molecular chaperones to guide conformational states of client proteins is essential to maintaining the health of cells and protecting them from various acute and chronic stress conditions. As a result, HSR activation may provide therapeutic benefit to certain types of tissue trauma (e.g. brain and heart ischemia) and neurodegenerative disorders (e.g. Huntington disease, Alzheimer disease, and Parkinson disease). Conversely, since aberrant expression of chaperones has been associated with tumorigenesis, compounds that down-regulate the HSR and chaperone levels could provide useful tools for combating cancer. To generate an effective readout for interrogating the HSR pathway, we engineered HeLa cervical cancer cells with an HSE driving beta-lactamase reporter gene expression (HSE-bla). A stably integrated pool of heat shock responsive cells was isolated by FACS and further evaluated using an inhibitor of Hsp90, 17-AAG. Hsp90 inhibition is known to upregulate HSF-1 activity leading to potent induction of the HSR, which can be readout using HSE-bla HeLa cells.

CellSensor™ NFAT HEK 293T DA Assay Kit Thermo Scientific™

The CellSensor® NFAT HEK 293T DA (Division Arrested) cells contain 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 a fixed concentration of PMA. 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 or these cells can be used in other assays where a sensitive readout to intracellular changes in calcium are needed.

CellSensor™ NFAT bla HEK 293T Cell Line Thermo Scientific™

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.

CellSensor™ NFKB-bla Jurkat Cell Line Thermo Scientific™

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™ c-Fos-bla ME-180 Cell Line Thermo Scientific™

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™ LEF/TCF-bla HCT-116 Cell Line Thermo Scientific™

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.

CellSensor™ CRE-bla Jurkat Cell Line Thermo Scientific™

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™ SIE-bla ME-180 Cell Line Thermo Scientific™

The CellSensor® SIE-bla ME-180 cell line contains a beta-lactamase reporter gene under control of the Sis-Inducible Element (SIE) stably integrated into ME-180 cells. To construct this cell line, the SIE-bla construct was transduced into ME-180 cells by lentivirus. Flow cytometry was used to isolate cells responsive to Interleukin-6 (IL-6). This cell line is validated for DMSO tolerance, cell number, stimulation time, substrate loading time, as well as Z' and EC50 concentrations of IL-6. The CellSensor® SIE-bla ME-180 cell line is responsive to IL-6, OSM and IFNγ 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 also be tested for dose response against this cell line. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ STAT6-bla RA-1 Cell Line Thermo Scientific™

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.

CellSensor™ AP-1-bla HEK293T Cell Line Thermo Scientific™

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.
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