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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™ 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™ 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™ 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™ 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.

LanthaScreen™ ERK2 A375 Cell Line

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

CellSensor™ CRE-bla 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™ SBE-bla A375 Cell Line

Bone morphogenetic proteins (BMPs), members of the transforming growth factor beta (TGF-β) superfamily, play important roles in the development of the heart, central nervous system and cartilage. Disruption of BMP signaling affects the body plan of the developing embryo. BMPs propagate their signal by activating Activin receptor-like kinase (ALK), which in turn mediates the phosphorylation of R-Smads. Phosphorylated R-Smad associates with Smad4 and then translocates to the nucleus regulating gene expression. A375 cells are human malignant melanoma cells containing endogenous Smad signaling pathway. SBE-bla A375 cell line was engineered to express beta-lactamase under the control of Smad binding element. This cell line has been validated for Z' and EC50 under optimized conditions using BMP-4. This cell line has also responded to Nodal and was tested for assay performance under variable conditions, including DMSO concentration, cell number, stimulation time, substrate loading time. Additional information using various small molecule inhibitors and StealthTM RNAi is also provided.

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™ 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™ 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™ 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.

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™ 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™ TrkB-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 (e.g., MAPK, PI3K, PKC).

BDNF (brain-derived neurotrophic factor) and its receptor TrkB (also known as NTRK2) play a fundamental role in regulating neural development, survival, and synaptic activity and plasticity. TrkB is also a potential anticancer target, since altered signaling through TrkB promotes tumor formation, survival, and metastasis of various cancers (including neuroblastomas, multiple myelomas, and pancreatic ductal adenocarcinomas). Moreover, growing evidence indicates that BDNF and its receptor influences food intake and body weight control.

CellSensor® TrkB-NFAT-bla CHO-K1 cells contain a beta-lactamase reporter gene under control of the NFAT Response Element that has been stably integrated into CHO-K1 cells along with TrkB. CellSensor® TrkB-NFAT-bla CHO-K1 cells express beta-lactamase upon stimulation with brain-derived neurotrophic factor (BDNF). This cell line is a clonal population isolated by flow cytometry and has been tested for robust performance by assessing a variety of assay parameters.