Shop All Pathway Analysis Cell Based Assay Kits

CellSensor™ NFkB-bla ME-180 Cell Line

The CellSensor® NFκB-bla ME-180 cell line contains a beta-lactamase reporter gene under control of the Nuclear Factor Kappa B (NFκB) response element stably integrated into ME-180 cells. The cell line was created through fluorescence activated cell sorting (FACS) of cells responsive to stimulation of the NFκB pathway with tumor necrosis factor alpha (TNFα). The cell line was validated for DMSO tolerance, incubation time with stimulant, and substrate loading conditions. The CellSensor® NFκB-bla ME-180 cell line responds to agonist treatment as expected from literature and can be adapted for high-throughput screening for agonists or antagonists of the NFκB 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™ 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™ 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™ 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.

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™ 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™ HSE-bla HeLa Cell Line

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.

LanthaScreen™ ERK2 U2OS 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). LanthaScreenTM Erk2 U2OS is a human cell line which constitutively expresses GFP-Erk2. The MapK signaling pathway is functionally intact in this cell line, therefore the GFP-Erk2 fusion protein serves as a substrate for the growth-factor-inducible phosphorylation by MEK. 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. GFP-Erk2 lentivirus was transduced into U2OS 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™ 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™ 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.

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™ 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™ 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™ 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.

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.
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