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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™ 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™ NFAT HEK 293T DA Assay Kit

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 CHO-K1 DA Assay Kit

The CellSensor® NFAT CHO-K1 DA (Division Arrested) cells contain a beta-lactamase (bla) reporter gene under control of a Nuclear Factor of Activated T Cells (NFAT) response element stably integrated into CHO-K1 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. These cells can also be used in other assays where a sensitive readout to intracellular changes in calcium 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™ 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.

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™ 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 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.

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™ NFKB-bla HEK293T Cell Line

The CellSensor® NFκB-bla HEK 293T cell line contains a beta-lactamase reporter gene under the control of the Nuclear Factor kappa B (NFκB) response element. To obtain the cell line, the NFκB-bla construct was transduced into HEK 293T cells by lentivirus. Subsequent flow cytometry was used to isolate clones responsive to Tumor Necrosis Factor alpha (TNFα). The cell line has been validated for DMSO tolerance, stimulation time, substrate loading time, and EC50 concentration of TNF&alpha. This cell line is responsive to TNFα and can be used to probe NFκB signaling pathways, including those that are involved in apoptosis, viral defense, cancer, inflammation, and autoimmune disease. 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™ 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™ NFAT-bla RA-1 Cell Line

B cell receptor (BCR)-mediated signaling pathways are important for B cell proliferation and differentiation. Abnormal B cell signaling has been linked to various diseases such as lupus, lymphoma, and various immune disorders. Antigen binding to the BCR promotes the activation of several protein tyrosine kinases (PTK), which leads to phosphorylation of the BCR complex and recruitment and activation of the PTK Syk. This in turn promotes phosphorylation of PLCγ, Shc, and Vav. Additionally, the Tec family member Btk is recruited to the plasma membrane, where it is involved in activation of PLCγ. Initiation of B lymphocyte activation is dependent on the tyrosine phosphorylation-dependent formation of multimolecular effector protein complexes that activate downstream signaling pathways including PKC/Ca2+/NFAT. This cell-based assay has been thoroughly tested and validated by Invitrogen and is suitable for immediate use in screening applications. The following information illustrates the comprehensive assay testing completed and validation of assay performance under optimized conditions (Figure 1).