Shop All Pathway Analysis Cell Based Assay Kits

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™ 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.

BacMam Akt [pSer473] 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 various substrates including AKT. BacMam provides a convenient genetic delivery tool for a GFP-AKT fusion protein in the cell line of interest. This kit describes an HTS-compatible cellular immunoassay measuring phosphorylation of GFP-AKT at Ser473.

BacMam Akt [pThr308] 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 various substrates including AKT. BacMam provides a convenient genetic delivery tool for a GFP-AKT fusion protein in the cell line of interest. This kit describes an HTS-compatible cellular immunoassay measuring phosphorylation of GFP-AKT at Thr308.

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™ 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™ SIE-bla ME-180 Cell Line

The CellSensor® SIE-bla ME-180 cell line contains a beta-lactamase reporter gene under control of the Sis-Inducible Element (SIE) stably integrated into ME-180 cells. To construct this cell line, the SIE-bla construct was transduced into ME-180 cells by lentivirus. Flow cytometry was used to isolate cells responsive to Interleukin-6 (IL-6). This cell line is validated for DMSO tolerance, cell number, stimulation time, substrate loading time, as well as Z' and EC50 concentrations of IL-6. The CellSensor® SIE-bla ME-180 cell line is responsive to IL-6, OSM and IFNγ 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 also be tested for dose response against this cell line. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ irf1-bla TF-1 Cell Line

Jak/Stat signaling pathways play essential roles in cellular responses to distinct cytokines. One of the 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 knockout causes embryonic lethality due to defective erythropoiesis, suggesting that the Jak2/Stat5 pathway plays an important role in red blood cell formation. The recent discovery of an activating mutation in JAK2 (V617F) present in a high percentage of myeloproliferative disease (MPD) patients suggests that the Jak2/Stat5 pathway may be a potential therapeutic target for certain forms of MPD. The activated Stat5 transcription factor recognizes and binds 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 TF1 Cell Line contains a beta - lactamase reporter gene under control of the irf - 1 response element stably integrated into TF1 cells. TF1 cells are a human erythroleukemia cell line that is growth - dependent on GM - CSF and have an intact GM - CSF - JAK2 - STAT5 pathway. This cell line is validated for EC50 and Z’ - factor using GM - CSF. This cell line has also been tested under variable experimental conditions, including DMSO concentration, cell number, stimulation time, and substrate loading time. Responsiveness to EPO and Jak Inhibitor 1 was also tested (Figures 2 and 3). CellSensor® irf1 - bla TF1 cells were stimulated in triplicate with GM - CSF over the indicated concentration range in a 384 - well format. Cells were incubated for 5 hrs with agonist and 0.5% DMSO and then combined with LiveBLAzer™ - FRET B/G Substrate (CCF4 - AM) for 2.5 hrs. Fluorescence emission values at 460 nm and 530 nm were obtained using a standard fluorescence plate reader. The 460/530 ratios were plotted for each replicate against the indicated concentrations of GM - CSF. CellSensor® irf1 - bla TF1 cells were stimulated with Epo over the indicated concentration range in a 384 - well format. Cells were incubated for 5 hrs with agonist and 0.5% DMSO and then combined with LiveBLAzer™ - FRET B/G Substrate (CCF4 - AM) for 2.5 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 Epo. CellSensor® irf1 - bla TF1 cells were treated with Jak Inhibitor 1 over the indicated concentration range in a 384 - well format. Cells were then stimulated with GM - CSF or EPO for 5 hrs in 0.5% DMSO and then combined with LiveBLAzer™ - FRET B/G Substrate (CCF4 - AM) for 2.5 hrs. Fluorescence emission values at 460 nm and 530 nm were obtained using a standard fluorescence plate reader. These values were converted to percent inhibition relative to a set of controls (unstimulated cells and EC80 of GM - CSF or EPO treated cells) and plotted against the indicated concentrations of Jak Inhibitor 1. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ SIE-bla HEK293T Cell Line

The CellSensor® SIE-bla HEK 293T cell line contains a beta-lactamase reporter gene under control of the SIE response element stably integrated into HEK 293T cells. The cell line was created through sorting by FACS of cells responsive to stimulation of the SIE pathway with interleukin 6 (IL-6). The cell line was validated for DMSO tolerance, incubation time with stimulant, and substrate loading conditions. The CellSensor® SIE-bla HEK 293T cell line responds to agonist treatment as expected from literature and can be adapted for high throughput screening for agonists or antagonists of the SIE 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.

LanthaScreen™ STAT3 GripTite™ Cell Line

The JAK/STAT3 signaling pathway is known to be activated by cytokines such as IL-6. In this pathway, binding of IL-6 to its cell-surface receptors results in the activation of JAKs, which in turn phosphoactivate STAT3 proteins at a specific tyrosine residue (Y705). LanthaScreen® STAT3 GripTite™ is a human cell line that constitutively expresses a GFP-STAT3 fusion protein. The JAK/STAT signaling pathway is known to be functionally intact in this cell line; therefore, the GFP-STAT3 fusion protein serves as a substrate for IL-6-inducible phosphorylation. Using this cell line, a lytic immunoassay has been developed in which the phosphorylation state of GFP-STAT3 is detected in cell lysates using a terbium-labeled anti-pY705-STAT3 antibody in a time-resolved FRET (TR-FRET) readout.

The GFP-STAT3 DNA expression construct was transfected into the GripTite™ HEK 293 cell line using Lipofectamine™ 2000 transfection reagent, and the transfected cells were selected with blasticidin. This cell line is a clonal population isolated by flow cytometry using GFP fluorescence as a sorting marker. The assay utilizing this cell line is validated for EC50 and Z’-factors under optimized conditions using IL-6 as a ligand for JAK-mediated GFP-STAT3 phosphorylation. This assay has also been tested under variable experimental conditions, including cell plating density, stimulation time, DMSO tolerance, and assay equilibration time. An alternate "mix-and-read" assay format is also described.

CellSensor™ AP-1-bla HEK293T Cell Line

The CellSensor® AP-1-bla HEK 293T Cell Line contains a beta-lactamase reporter gene under control of the AP-1 response element stably integrated into HEK 293T cells. The cell line was created through sorting by FACS of cells responsive to stimulation of the AP-1 pathway with phorbol 12-myristate 13-acetate (PMA). The cell line was validated for DMSO tolerance, incubation time with stimulant, and substrate loading conditions. The AP-1-bla HEK 293T cell line responds to agonist treatment as expected from 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™ 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.

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™ NFκB-bla RA 1 Cell Line

The CellSensor® NFκB-bla RA-1 cell line contains a beta-lactamase reporter gene under control of the NFκB response element stably integrated into Ramos 1 (RA-1) cells. To construct this cell line, the NFκB -bla construct was transduced into RA-1 cells by lentivirus. Flow cytometry was used to isolate cells responsive to Tumor Necrosis Factor (TNFThis cell line was validated for DMSO tolerance, cell number, stimulation time, substrate loading time, as well as Z´ and EC50 concentrations for TNF. The CellSensor® NFκB-bla RA-1 cell line is responsive to TNFand can be used to probe NFκB signaling pathways, which are involved in the regulation of apoptosis, viral replication, tumorigenesis, inflammation, and various autoimmune diseases. 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™ 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.