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GeneBLAzer™ CRHR2-CRE-bla CHO-K1 Cells Thermo Scientific™

GeneBLAzer® CRHR2-CRE-bla CHO-K1 cells contain the human Corticotropin Releasing Factor Receptor 2 (CRHR2) stably integrated into the CellSensor® CRE-bla CHO-K1 cell line. CellSensor® CRE-bla CHO-K1 cells (Cat. no. K1535) contain a beta-lactamase reporter gene under control of the Cyclic AMP Response Element (CRE) response element.

The GeneBLAzer® CRHR2-CRE-bla CHO-K1 cells are functionally validated for Z' and EC50 concentrations of CRF. In addition, GeneBLAzer® CRHR2-CRE-bla CHO-K1 cells have been tested for assay performance under variable conditions. These data are found in the Validation & Assay Performance Summary.

Corticotropin releasing factor is a 41-amino acid peptide that plays a role in the integration of autonomic, neuroendocrine, and behavioral responses to stress. These effects are mediated through two receptor families, CRHR1 and CRHR2. While CRF was originally isolated from the hypothalamus, where it was shown to be the primary neuroregulator mediating the hypothalamic-pituitary-adrenocortical stress axis, it has since been found to be widely distributed outside the hypothalamus throughout the central nervous system. Presently, there are five distinct targets for CRF with unique pharmacology and localization. These have been placed into three distinct classes, two of which are the G-protein-coupled receptors CRF1 (CRHR1) and CRF2 (CRHR2). Three functional splice variants have been identified for the mammalian CRHR2 receptor, although pharmacological characterization of these splice variants revealed no major differences between them.

For Academic pricing please login or contact us at discoverysciences@invitrogen.com.

GeneBLAzer™ BDKRB1-NFAT-bla CHO-K1 Cells Thermo Scientific™

GeneBLAzer® BDKRB1-NFAT-bla CHO-K1 cells contain the human Bradykinin Receptor 1 (BDKBR1) stably integrated into the CellSensor® NFAT-bla CHO-K1 cell line. CellSensor® NFAT-bla CHO-K1 cells (Cat. no. K1534) contain a beta-lactamase reporter gene under control of the Nuclear Factor of Activated T-cells (NFAT) response element.

The GeneBLAzer® BDKRB1-NFAT-bla CHO-K1 cells are functionally validated for Z' and EC50 concentrations of [Lys-des-Arg9]-Bradykinin. In addition, GeneBLAzer® BDKRB1-NFAT-bla CHO-K1 cells have been tested for assay performance under variable conditions. These data are found in the Validation & Assay Performance Summary.

The Bradykinin-BDKRB1 receptor has been implicated in inflammation and pain. Preclinical evidence demonstrates that interruption of BDKRB1 receptor function causes analgesia under a variety of conditions and has been well established using selective antagonists and genetically modified mice.

For Academic pricing please login or contact us at discoverysciences@invitrogen.com.

Chromatin Condensation & Membrane Permeability Dead Cell Apoptosis Kit with Hoechst 33342, YO-PRO™-1, and PI dyes, for flow cytometry Invitrogen™

This product detects apoptotic cells with changes in nuclear chromatin condensation and plasma membrane permeability, using three nucleic acid stains: UV-excitable Hoechst 33342, green fluorescent YO-PRO™ dye, and propidium iodide. The YO-PRO™ dye can enter apoptotic cells, whereas red-fluorescent propidium iodide (PI) cannot. Thus after staining with YO-PRO™-1 dye and PI, apoptotic cells show green fluorescence, and dead cells show primarily red fluorescence and some green fluorescence. Blue fluorescent Hoechst 33342 brightly stains the condensed chromatin of apoptotic cells and more dimly stains the normal chromatin of live cells. The staining pattern resulting from the simultaneous use of these three dyes makes it possible to distinguish normal, apoptotic and dead cell populations by flow cytometry or fluorescence microscopy.

View a selection guide for all apoptosis assays for flow cytometry.

GeneBLAzer™ M1-NFAT-bla CHO-K1 Cells Thermo Scientific™

GeneBLAzer® M1-NFAT-bla CHO-K1 cells contain the human Acetylcholine (muscarinic) subtype 1 receptor (M1) stably integrated into the CellSensor® NFAT-bla CHO-K1 cell line. CellSensor® NFAT-bla CHO-K1 cells (Cat. no. K1534) contain a beta-lactamase (bla) reporter gene under control of the Nuclear Factor of Activated T-cells (NFAT) response element.

M1-NFAT-bla CHO-K1 cells are functionally validated for Z'-factor and EC50 concentrations of carbachol. In addition, M1-NFAT-bla CHO-K1 cells have been tested for assay performance under variable conditions. These data are found in the Validation & Assay Performance Summary.

In the brain, M1 activation mediates 'slow' neuronal excitability. Cortical and hippocampal muscarinic receptors are thought to be important in the attentional aspects of cognition. The predominant receptor subtypes in these brain areas are M1, M3, and M4. Therefore, M1 is a potential target for cognition, Alzheimer's, dementia, and schizophrenia. Studies on knock-out mouse models of M1 are also beginning to reveal potential functions of the receptor. Additional information on the muscarinic receptors can be found in reviews.

For Academic pricing please login or contact us at discoverysciences@invitrogen.com.

MANT-GTP (2'-(or-3')-O-(N-Methylanthraniloyl) Guanosine 5'-Triphosphate, Trisodium Salt) Invitrogen™

The nucleotide analog MANT GTP is modified on the ribose moiety. The compact mature of the MANT fluorophore and its attachment position results in nucleotide analogs that induce minimal perturbation of nucleotide-protein interactions. Because MANT fluorescence is sensitive to the environment of the fluorophore, nucleotide-protein interactions may be directly detectable. MANT nucleotides are valuable probes of the structure and enzymatic activity of nucleotide-binding proteins.

GeneBLAzer™ MR DA Assay Kit Thermo Scientific™

GeneBLAzer®MR DA(Division Arrested) cells and MR-UAS-bla HEK 293T cells contain the ligand-binding domain (LBD) of the human Mineralcorticoid Receptor (MR) fused to the NAbinding domain of GAL4 stably integrated in the GeneBLAzer®UAS-bla HEK 293T cell line. GeneBLAzer®UAS-bla HEK 293T cells stably express a beta-lactamase reporter gene under the transcriptional control of an upstream activator sequence (UAS). When an agonist binds to the LBD of the GAL4 (DBD)-MR (LBD) fusion protein, the protein binds to the UAS, resulting in expression of beta-lactamase. Division Arrested (DA) cells are available in two configurations- an Assay Kit (which includes cells and sufficient substrate to analyze 1 x 384- well plate), and a tube of cells sufficient to analyze 10 x 384-well plates. 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. Both MR DA cells and MRUAS-bla HEK 293T cells are functionally validated for Z' and EC50 concentrations of aldosterone. In addition, MR-UAS-bla HEK 293T cells have been tested for assay performance under variable conditions, including DMSO concentration, cell number, and substrate loading time. Additional testing data using alternate stimuli are also available.

CellSensor™ c-Fos-bla ME-180 Cell Line Thermo Scientific™

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.

Tango™ AGTRL1-bla U2OS Cells Thermo Scientific™

The Tango™ AGTRL1-bla U2OS cells contain the human Angtiotension-Like Receptor 1 (AGTRL1) linked to a TEV protease site and a Gal4-VP16 transcription factor stably integrated into the Tango™ GPCR-bla U2OS parental cell line. This parental cell line stably expresses a beta-arrestin⁄TEV protease fusion protein and the beta-lactamase (bla) reporter gene under the control of a UAS response element.

The Tango™ AGTRL1-bla U2OS cells have been functionally validated for Z' factor and EC50 concentrations of established ligands. In addition, Tango™AGTRL1-bla U2OS cells have been tested for assay performance under variable conditions. These data are found in the Validation & Assay Performance Summary for each product.

GeneBLAzer™ CRHR1-CRE-blaCHO-K1 Cells Thermo Scientific™

GeneBLAzer® CRHR1-CRE-bla CHO-K1 cells contain the human Corticotropin Releasing Factor Receptor 1 (CRHR1) stably integrated into the CellSensor® CRE-bla CHO-K1 cell line. CellSensor® CRE-bla CHO-K1 cells (Cat. no. K1535) contain a beta-lactamase reporter gene under control of the Cyclic AMP Response Element (CRE) response element.

The GeneBLAzer® CRHR1-CRE-bla CHO-K1 cells are functionally validated for Z' and EC50 concentrations of CRF. In addition, GeneBLAzer® CRHR1-CRE-bla CHO-K1 cells have been tested for assay performance under variable conditions. These data are found in the Validation & Assay Performance Summary.

Corticotropin releasing factor is a 41-amino acid peptide that plays a role in the integration of autonomic, neuroendocrine, and behavioral responses to stress. These effects are mediated through two receptor families, CRHR1 and CRHR2. While CRF was originally isolated from the hypothalamus, where it was shown to be the primary neuroregulator mediating the hypothalamic-pituitary-adrenocortical stress axis, it has since been found to be widely distributed outside the hypothalamus throughout the central nervous system. Presently, there are five distinct targets for CRF with unique pharmacology and localization. These have been placed into three distinct classes, two of which are the G-protein-coupled receptors CRF1 (CRHR1) and CRF2 (CRHR2). Three functional splice variants have been identified for the mammalian CRHR2 receptor, although pharmacological characterization of these splice variants revealed no major differences between them.

For Academic pricing please login or contact us at discoverysciences@invitrogen.com.

eBioscience™ Annexin V Apoptosis Detection Kit PE Invitrogen™

Annexins are a family of calcium-dependent phospholipid-binding proteins that preferentially bind phosphatidylserine (PS). Under normal physiologic conditions, PS is predominantly located in the inner leaflet of the plasma membrane. Upon initiation of apoptosis, PS loses its asymmetric distribution across the phospholipid bilayer and is translocated to the extracellular membrane leaflet marking cells as targets of phagocytosis. Once on the outer surface of the membrane, PS can be detected by fluorescently labeled Annexin V in a calcium-dependent manner.

In early-stage apoptosis, the plasma membrane excludes viability dyes such as propidium iodide (PI), 7-AAD, or Fixable Viability Dyes such as eFluor™ 660 or eFluor™ 780. These cells will stain with Annexin V but not a viability dye, thus distinguishing cells in early apoptosis. However, in late stage apoptosis, the cell membrane loses integrity thereby allowing Annexin V to also access PS in the interior of the cell. A viability dye can be used to resolve these late-stage apoptotic and necrotic cells (Annexin V, viability dye-positive) from the early-stage apoptotic cells (Annexin V positive, viability dye-negative).

Note: Fixable Viability Dye eFluor™ 450 is not recommended for use with Annexin V Apoptosis Detection Kits.

Reported Application
Flow Cytometric Analysis

CellEvent™ Senescence Green Detection Kit Invitrogen™

The CellEvent Senescence Green Detection Kit consists of a fluorescent probe and optimized buffer that enable the image-based detection of senescent cells. The fluorescein-based probe contains two galactoside moieties, making it a target for β-galactosidase. Activation of β-galactosidase is commonly used as a biomarker for senescent cells. This hydrolase enzyme resides in lysosomes and converts β-galactosides into monosaccharides under acidic pH conditions. The enzymatically cleaved product is retained within the cell due to covalent binding of intracellular proteins and emits a fluorogenic signal that has absorption/emission maxima of 490/514 nm.

CellEvent Senescence Green Detection Kit features include:
• Reliable and quick fluorescent detection of senescent cells
• Greater sensitivity than traditional colorimetric x-gal
• Fluorescent signal is well retained and detected using standard Alexa Fluor 488/FITC filter sets
• Multiplex-enabled—fluorescent senescence probe can be multiplexed

Due to a limited replicative lifespan, normal cells enter cell cycle arrest, also known as cellular senescence. While in this senescent phase the cells remain metabolically active without undergoing cell death or division. They adopt a specific phenotypic that includes the appearance of multiple nuclei, increased vacuolization, expression of pH-dependent β-galactosidase, and morphological enlargement and extension. Senescence, through a variety of mechanisms, may play a role in tumor suppression, tumor progression, aging, and tissue repair.

Activation of β-galactosidase is commonly used as a biomarker for senescent cells. This hydrolase enzyme resides in lysosomes and converts β-galactosides into monosaccharides under acidic pH conditions. The activity is optimal at lysosomal pH 4, but conventional assays measure at pH 6. It has been shown that normalized β-galactosidase activity is twice as high in senescent cells as in pre-senescent cells regardless of the pH value used for testing.

A colorimetric substrate for β-galactosidase, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (x-gal), has long been used to detect metabolic activity in cells in vitro. However, its use is limited due to inconsistent signal, length of assay, and inability to multiplex.

Therefore, we have developed a sensitive, fluorescent substrate for β-galactosidase that can be used for the detection of senescent cells. The CellEvent Senescence Green probe is a fluorescein-based reagent that contains two galactoside moieties, making it a specific target of β-galactosidase. The enzyme- cleaved product is retained within the cell due to covalent binding of intracellular proteins and emits a fluorogenic signal that has absorption/emission maxima of 490/514 nm.

The CellEvent Senescent Green Kit provides CellEvent Senescent Green probe and an optimized buffer for the detection of senescent cells in fixed samples. This probe can be multiplexed with other fluorescent reagents compatible with paraformaldehyde fixation.

Fura-2, Pentasodium Salt, cell impermeant Invitrogen™

Fura-2, pentasodium salt is an intracellular calcium indicator that is ratiometric and UV light—excitable. This water-soluble salt form is useful for intracellular loading by microinjection, infusion from patch pipette or uptake induced by our Influx pinocytic cell-loading reagent (I-14402).

Learn more about ion indicators including calcium, potassium, pH, and membrane potential indicators ›

CaspGLOW™ Fluorescein Active Caspase-3 Staining Kit Invitrogen™

The CaspGLOW™ Fluorescein Active Caspase-3 Staining Kit contains the reagents to sensitively detect active Caspase-3 in mammalian cells. This assay utilizes the inhibitor specific for Caspase-3, DEVD-FMK, that is directly conjugated to FITC (fluorescein isothiocyanate) for the detection system. This reagent is cell permeable, non-toxic and irreversibly binds to the active enzyme. Detection of the labeled cells can be determined by flow cytometry, fluorescent microscopy or a fluorescent plate reader.

The Caspases are a family of aspartate-specific cysteine proteases that act in a step-wise signaling manner like kinases. Recruitment of these proteases to oligomerized receptors leads to activation and autoproteolytic cleavage. Active caspases can proteolyze additional caspases generating a caspase cascade. The final outcome of this signaling pathway is a form of controlled cell death, termed apoptosis.

Caspase-3, also known as Yama, CPP32, and apopain, cleaves its substrates at the carboxyl terminus of aspartate residues. Active Caspase-3 consists of 2 sets of homodimers (~ 17 and 12 kDa) that are derived from two precursor Caspase-3 polypeptides and has two active sites. Caspase-3 is proteolytically activated by other caspases.

Both subunits contribute to substrate binding and catalysis. The active site cysteine that covalently binds the substrate is located near the C-terminus of the large subunit. Active Caspase-3 has two-fold symmetry, two active site pockets each residing on an opposite side. Caspase-3, together with Caspases 8 and 9, is situated at pivotal junctions in apoptotic pathways. Caspase-3 appears to amplify Caspase 8 and 9 initiation signals into complete commitment to apoptotic disassembly.

Reported Application
Flow Cytometric Analysis, Immunocytochemistry

MANT-ADP (2'-(or-3')-O-(N-Methylanthraniloyl) Adenosine 5'-Diphosphate, Disodium Salt) Invitrogen™

The nucleotide analog MANT ADP is modified on the ribose moiety. The compact mature of the MANT fluorophore and its attachment position results in nucleotide analogs that induce minimal perturbation of nucleotide-protein interactions. Because MANT fluorescence is sensitive to the environment of the fluorophore, nucleotide-protein interactions may be directly detectable. MANT nucleotides are valuable probes of the structure and enzymatic activity of nucleotide-binding proteins.

Tango™ TACR1-bla U2OS Cells Thermo Scientific™

The Tango™ TACR1-bla U2OS cells contain the human Tachykinin 1 (TACR1) linked to a TEV protease site and a Gal4-VP16 transcription factor stably integrated into the Tango™ GPCR-bla U2OS parental cell line. This parental cell line stably expresses a beta-arrestin/TEV protease fusion protein and the beta-lactamase (bla) reporter gene under the control of a UAS response element.

The Tango™ TACR1-bla U2OS cells are functionally validated for Z' and EC50 concentrations of (SAR9,MET(02)11)-Substance P. In addition, Tango™ TACR1-bla U2OS cells have been tested for assay performance under variable conditions. These data are found in the Validation & Assay Performance Summary.

For Academic pricing please login or contact us at discoverysciences@invitrogen.com.
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