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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™ 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™ 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.

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™ 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™ 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™ NFKB-bla Freestyle™ HEK293F Cell Line

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

LanthaScreen™ STAT5 TF-1 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. In hematopoeitic cells, 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. In this pathway, binding of these cytokines to their respective cell surface receptors results in the activation of JAK2, which in turn phospho-activates STAT5 proteins at specific tyrosine residues (Tyr-694⁄699). LanthaScreenTM-STAT5 TF-1 is a human hematopoeitic cell line which constitutively expresses GFP-STAT5 fusion proteins. The JAK2⁄STAT5 signaling pathway is known to be functionally intact in this cell line, therefore the GFP-STAT5 fusion protein serves as a substrate for the inducible phosphorylation by JAK2. Using this cell line, a homogenous immuno-assay has been developed in which the phosphorylation state of GFP-STAT5 is detected in cell lysates using a LanthaScreenTM Terbium-anti-mouse and anti-phospho STAT5 [pTyr694⁄699] antibody pair, in a time-resolved FRET (TR-FRET) readout. GFP-STAT5 Lentivirus was transduced into TF-1 cells followed by selection with Blasticidine. 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 fusion protein. Using a lytic TR-FRET immuno-assay, this cell line is validated for EC50 and Z' under optimized conditions using GM-CSF as an agonist for JAK2 -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.

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™ 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™ ESRE-bla HeLa Cell Line

Endoplasmic Reticulum (ER) stress is associated with a variety of pathophysiological conditions, such as neurodegenerative diseases, diabetes, tumor growth under hypoxic conditions, and ischemic heart disease. Proteins in the ER misfold or unfold, and accumulate under stress conditions, which promote the expression of ER stress responsive genes. One of the mechanisms for mediating ER stress response is the activation of transcription factor ATF6. The quiescent form of ATF6 (p90ATF6), a type II-transmembrane protein, is embedded in the ER membrane and proteolyzed in an ER stress-dependent manner. The liberated N-terminal fragment (p50ATF6) translocates to the nucleus, binding to ER stress response element (ERSE) present in the proximal promoter regions of many ER stress-responsive proteins including ER chaperones. To better understand the pathological processes and provide novel avenues to potential therapies, ESRE–bla HeLa cells are engineered to express beta-lactamase under the control of ER stress response element. This is a clonal population isolated by FACS and its dose response curves with tunicamycin and thapsigargin are performed. This cell line also response to other known ER stress inducers.

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™ 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™ 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.