Shop All Pathway Analysis Cell Based Assay Kits

CellSensor™ TNFa-bla THP-1 Cell Line

Tumor necrosis factor-alpha (TNFα) cytokine cell signaling is a critical pathway involved in immune and inflammatory responses. In addition, TNFα has been shown to inhibit growth and induce differentiation in tumor and non-tumor cells. The CellSensor™ TNFα-bla THP-1 cell line is a suspension cell line derived from human acute monocytic leukemia cells. The TNFα-bla THP-1 cell line is used to measure the TNFα cytokine signaling pathway via transcriptional regulation of the beta-lactamase gene. This cell line was constructed using GenomeScreen™ technology utilizing a promoterless beta-lactamase reporter gene randomly integrated into the genome of the THP-1 cell line. Sequential rounds of sorting were performed to isolate cell clones containing tagged genes induced by TNFα. These cells have been shown to be responsive to TNFa agonist. 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.

LanthaScreen™ LRRK2 [pSer935] Cellular Assay Kit

The LanthaScreen® LRRK2 [pSer935] Cellular Assay Kit combines the flexibility of the BacMam gene delivery system with the robustness and power of our LanthaScreen® TR-FRET technology. It allows researchers to interrogate phosphorylation at serine 935 on a wild type Leucine-Rich Repeat Kinase-2 (LRRK2) protein in a variety of cell backgrounds to identify LRRK2 kinase inhibitors.

This assay format provides a convenient tool for the expression of LRRK2-GFP fusion protein in the cell background of of your choice. The resulting cellular assay can be used to identify LRRK2 and mutant inhibitorss, aiding in Parkinson's disease research and other research areas.

This kit provides enough material to evaluate the technology in a 384-well plate; larger sizes of all reagents are available separately.

The kit includes:

• LanthaScreen® Tb-anti-LRRK2 [pSer935] Antibody, 5 µg
• BacMam LRRK2-GFP Wild Type Reagent, 5 × 1 mL
• LanthaScreen® 6x Lysis Buffer, 6 mL
• Instrument Control Terbium TR-FRET Kit

A kit for interrogating a mutant LRRK2 protein is also available.

Benefits of this assay include:

More Physiologically Relevant: cell-based LRRK2 kinase activity and choice of cell background
More Convenient: compatible with High Throughput Screening (HTS); LanthaScreen® technology reduces interference and noise
Disease Relevant: screen compound libraries for Parkinson's disease-relevant molecules

More Physiologically Relevant
The LanthaScreen® Tb-Anti-LRRK2 [pSer935] Antibody allows the investigation of phosphorylation at serine 935 on LRRK2 protein kinase. When paired with LRRK2 BacMam reagents, you have the freedom to choose the cellular background for your assay, including primary cells. This enables screening for potential inhibitors of LRRK2 proteins in their natural complexes in a physiologically relevant cell type.

More Convenient
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 this assay ideal for HTS applications. The LanthaScreen® technology provides all of the advantages of TR-FRET ratiometric detection, including reduced data noise, less interference from fluorescent compounds, and high sensitivity, which results in the use of fewer cells than traditional Western or ELISA methods.

Disease Relevant
The LanthaScreen® Tb-Anti-LRRK2 [pSer935] Antibody can be used by Parkinson's and other disease researchers to perform HTS, cell-based assays to generate inhibition curves (IC50) for their compound libraries. This will aid drug discovery efforts by reducing the time to results from 4 hrs to typically 1.5 hrs compared to alternative ELISAs.

For Research Use Only. Not intended for animal or human diagnostic or therapeutic use.

Related Links:
LRRK2 tools for advancing Parkinson's disease research
Kinase protein portfolio
LanthaScreen® Eu Kinase Binding Assay
LanthaScreen® Activity Assay
Learn More About BacMam Technology

CellSensor™ NFkB-bla RAW 264.7 Cell Line

The CellSensor® NFκB-bla RAW264.7 mouse macrophage cell line contains a beta-lactamase reporter gene under control of a stably integrated NFκB response element. This cell line is a clonal population isolated in response to lipopolysaccharide by flow cytometry. This cell line has been tested for assay performance under variable conditions, including DMSO concentration, cell number, stimulation time, and substrate loading time, and has been validated for Zfi-factor and EC50 concentrations of the TLR7 ligand imiquimod. Additional data using alternate stimuli are shown. The molecules responsible for coordinating the immune systems recognition of pathogens are Toll-like receptors (TLRs), of which there are currently 10 identified members in humans. TLRs are pattern recognition receptors (PRRs), which bind to pathogen-associated molecular patterns (PAMPs), small molecular sequences consistently found on pathogens. This binding activates the transcription of immune system genes and regulators such as cytokines, chemokines, and costimulatory molecules, thereby promoting the initial immune response of macrophages and neutrophils. Academic and non-profit customers, please inquire for special pricing.

LanthaScreen™ LRRK2 G2019S [pSer935] Cellular Assay Kit

The LanthaScreen® LRRK2 G2019S [pSer935] Cellular Assay Kit combines the flexibility of the BacMam gene delivery system with the robustness and power of our LanthaScreen® TR-FRET technology. It allows researchers to interrogate phosphorylation at serine 935 on a disease-relevant mutant (G2019S) Leucine-Rich Repeat Kinase-2 (LRRK2) protein in a variety of cell backgrounds to identify LRRK2 kinase inhibitors.

This assay format provides a convenient tool for the expression of G2019S LRRK2-GFP fusion protein in the cell background of your choice. The resulting cellular assay can be used to identify LRRK2 G2019S inhibitors, aiding in Parkinson's disease and other research focuses.

This kit provides enough material to evaluate the technology in a 384-well plate; larger sizes of all reagents are available separately.

The kit includes:

• LanthaScreen® Tb-anti-LRRK2 [pSer935] Antibody, 5 µg
• BacMam LRRK2-GFP G2019S Reagent, 5 × 1 mL
• LanthaScreen® 6x Lysis Buffer, 6 mL
• Instrument Control Terbium TR-FRET kit

A kit for interrogating wild-type LRRK2 protein is also available.

Benefits of this assay include:

More Physiologically Relevant: cell-based LRRK2 kinase activity and choice of cell background
More Convenient: compatible with High Throughput Screening (HTS); LanthaScreen® technology reduces interference and noise
Disease Relevant: screen compound libraries for Parkinson's disease-relevant molecules

More Physiologically Relevant
The LanthaScreen® Tb-Anti-LRRK2 [pSer935] Antibody allows the investigation of phosphorylation at serine 935 on LRRK2 protein kinase. When paired with LRRK2 BacMam reagents, you have the freedom to choose the cellular background for your assay, including primary cells. This enables screening for potential inhibitors of LRRK2 proteins in their natural complexes in a physiologically relevant cell type.

More Convenient
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 this assay ideal for HTS applications. The LanthaScreen® technology provides all of the advantages of TR-FRET ratiometric detection, including reduced data noise, less interference from fluorescent compounds, and high sensitivity, which results in the use of fewer cells than traditional Western or ELISA methods.

Disease Relevant
The LanthaScreen® Tb-Anti-LRRK2 [pSer935] Antibody can be used by Parkinson's and other disease researchers to perform HTS, cell-based assays to generate inhibition curves (IC50) for their compound libraries. This will aid drug discovery efforts by reducing the time to results from 4 hrs to typically 1.5 hrs compared to alternative ELISAs.

For Research Use Only. Not intended for animal or human diagnostic or therapeutic use.

Related Links:
LRRK2 tools for advancing Parkinson's disease research
Kinase protein portfolio
LanthaScreen® Eu Kinase Binding Assay
LanthaScreen® Activity Assay
Learn More About BacMam Technology

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™ irf1-bla HEL Cell Line

Jak/Stat signaling pathways play essential roles in the cellular responses to distinct cytokines. One of 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 knock-out causes embryonic lethality due to defective erythropoiesis, suggesting the Jak2/Stat5 pathway plays important role in red blood cell formation. Recent discovery of activating mutation in JAK2 (V617F) present in high percentage of myeloproliferative disease (MPD) patients suggests Jak2/Stat5 pathway to be the potential therapeutic target for certain forms of MPD. The activated transcription factor Stat5 dimers recognize and bind 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 HEL cell line contains a beta-lactamase reporter gene under control of the interferon regulatory factor-1 (irf1) response element stably integrated into HEL cells. HEL cells are a human erythroleukemia cell line that is growth factor independent and contains a endogenous homozygous JAK2V617F mutation. This cell line validated for IC50 and Z'-Factor under optimized conditions using Jak Inhibitor 1 (Figure 1). This cell line has also been tested under variable 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 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™ 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 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.

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™ AP-1-bla A375 Cell Line

The CellSensor® AP1-bla A375 cell line contains a beta-lactamase reporter gene under control of the AP1 response element stably integrated into A375 cells. A375 cells are human melanoma cancer cells that contain the endogenous B-Raf mutation V600E resulting in constitutive B-Raf kinase activity. The CellSensor® AP1-bla A375 cell line is a clonal population isolated by flow cytometry based on constitutive expression of beta-lactamase. This cell line has been validated with various small-molecule Raf inhibitors as well as B-Raf Stealth RNAi™ siRNA. 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 Zfi-factor and IC50 concentrations of Raf1 Inhibitor I. The RAF gene family (RAF1, A-RAF, and B-RAF) encodes closely related serine/threonine protein kinases that are important effectors of Ras activation. Raf1 and A-Raf are rarely mutated, whereas mutations in B-Raf are common in human cancers, especially melanoma. B-Raf is mutated in about 70% of human melanomas, 35-70% of papillary thyroid carcinomas, and less commonly in lung and colorectal carcinomas. Mutations are mostly in the B-Raf kinase domain and, in melanomas, the vast majority are V600E missense mutations leading to activation of B-Raf kinase. The constitutive activity of B-Raf V600E can directly lead to the activation of the Mek/MAPK signaling pathway. Therefore, inhibition of B-Raf/Mek/MAPK signaling could be a potential way of treating melanomas and other tumors with mutant B-Raf.

CellSensor™ Myc-bla HCT116 Cell Line

The CellSensor™Myc-bla HCT116 cell line contains a beta-lactamase reporter gene under the control of Myc binding sequences. The construct was transduced into HCT116 cells using a lentiviral system. HCT116 is a colon cancer cell line which expresses a mutated form of beta-catenin. This form of beta-catenin leads to the accumulation of beta-catenin and constitutive activation of downstream genes such as Myc. This cell line is a clonal population isolated by flow cytometry. It has been validated for cell plating density and DMSO tolerance. The signaling pathway has been validated using RNAi against c-Myc and ICG-001, an inhibitor of the wnt-beta-catenin pathway. The expression of the mutant beta-catenin in HCT116 cells results in constitutive activation of beta-lactamase in this CellSensor™line, which can be knocked down by ICG-001 (Figure 1) or Myc RNAi (Figure 2).

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.

GeneBLAzer™ Jurkat Control Kit

Th e GeneBLAzer® Jurkat Control Kit contains constitutively expressing (CMV-bla) and wildtype Jurkat cells to provide positive and negative controls, respectively, for evaluating beta-lactamase activity (Figure 1).