Innovative Tools for Molecular and Cell Biology

Innovative Tools for Molecular and Cell Biology

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Fluorescent Biosensors for Live Cell Imaging

Bright Red and Green Fluorescent Sensors

Simultaneous measurements of GPCR signaling in live HEK 293 cells. Green sensors in the top panels, red sensors in the bottom panels. M1 muscarinic receptor activated by carbachol.

Red DAG sensor multiplexed with a green cAMP sensor indicates dose-dependent signaling via calcitocin receptor

Red DAG sensor multiplexed with a green cAMP sensor indicates dose-dependent signaling via calcitocin receptor.

Montana Molecular Assay Steps Diagram

Montana Molecular Assay Steps Diagram

Features

  • Easy assay steps – No cell lysis
  • BacMam delivery to most cell types
  • Fluorescence imaging or plate reader detection (Z’ > 0.8)
  • Physiologically relevant and cell friendly
  • No forskolin or IBMX needed
  • No enzymes or co-factors
  • Multiplex GPCR Assays: Gs, Gq, Gi, Voltage

Description

  • While fluorescent dye-based assays are powerful tools for high-throughput screening in engineered cell lines or cell lysates to show that signaling has occurred, genetically encoded sensors that measure dynamic processes in physiologically relevant cells provide a higher level of insight into how signals are regulated in both space and time. It is the regulation of signaling that controls cell function in both health and disease.
  • The fluorescent assays for cAMP, DAG, and PIP2 described here are compatible with both GPCR drug discovery and basic research. Each sensor is packaged in a BacMam vector for efficient delivery to a wide variety of cell types, including cardiomyocytes, neurons, fibroblasts, pancreatic islet cells, and osteoblasts as well as standard cell lines such as HEK293, CHO, and NIH3T3.
  • BacMam as a delivery vector was originally described by Boyce and Bucher in 1996. In mammalian cells, this vector, based on a baculovirus, drives strong and exclusive expression of the gene encoding the fluorescent biosensor. The amount of virus introduced into a well can be easily adjusted, so you can control the transduction efficiency and expression levels (Kost et al, 2007).

How Do these Live Cell Assays Work?

  • The principle of the assays is essentially based on single Red and Green Fluorescent Protein sensors, meaning the genetic information for either GFP or RFP linked to an analyte specific sensor protein domain is introduced into the cells to be investigated. Upon analyte binding to the sensor, the fluorescence intensity of GFP or RFP changes which can be easily detected.
  •  1. Triggering of membrane-associated receptors by external signals produces second messengers: DAG, cAMP, PIP2, Ca2+
  •  2. Second messenger binds the biosensor
  •  3. Biosensor changes conformation
  •  4. Conformation change increases (or decreases) fluorescence intensity
  • The genetic transfer is done with BacMam vectors based on a modified baculovirus system. In mammalian cells, the baculovirus genome is silent, and it cannot replicate to produce new virus in mammalian cells. For the workflow of the assay see picture below.

Kits are Available for Live Cell Measurements of Various Cellular Parameters:

  •  • cAMP
  •  • DAG
  •  • PIP2
  •  • Ca2+
  •  • Voltage changes
  • The sensors are partly available with either GFP or RFP and with different promotors optimized for different target cells (ArcLight Voltage Sensor either with CMV or synapsin promoters). The availability of most of the sensors in 2 colors (GFP and RFP) allows for parallel measurements of different second messengers (multiplexing).

Product Overview

ORDER INFORMATION

  • For shipping and storage information please click on Order#.

cADDis cAMP Biosensor Assay Kits

Order# Description Amount Price Data Sheet
D0200G-10 Green Downward cAMP in BacMam 10 ml Kit 687,00 PDF
D0200G-30 Green Downward cAMP in BacMam 30 ml Kit 1379,00 PDF
D0200G-60 Green Downward cAMP in BacMam 60 ml Kit 2071,00 PDF
U0200G-10 Green Upward cAMP in BacMam 10 ml Kit 687,00 PDF
U0200G-30 Green Upward cAMP in BacMam 30 ml Kit 1379,00 PDF
U0200G-60 Green Upward cAMP in BacMam 60 ml Kit 2071,00 PDF
X0200G-5 Green Gi cADDis cAMP - Trial Kit 5 ml 456,00 PDF
X0200G-10 Green Gi cADDis cAMP in BacMam 10 ml Kit 687,00 PDF
X0200G-30 Green Gi cADDis cAMP in BacMam 30 ml Kit 1379,00 PDF
X0200G-60 Green Gi cADDis cAMP 60 ml Kit 2071,00 PDF
U0200R-10 Red Upward cAMP in BacMam 10 ml Kit 687,00 PDF
D0201G-10 Green Cilia-Targeted cAMP in BacMam 10 ml Kit 687,00 PDF
D0202G-10 Synapsin promoter Green Downward cAMP in BacMam 10 ml Kit 721,00 PDF
U0202G-10 Synapsin promoter Green Upward cAMP in BacMam 10 ml Kit 721,00 PDF
  • All prices are in EURO excl. VAT and shipping. Only available in Europe.

Diacylglycerol (DAG) Biosensor Assay Kits

Order# Description Amount Price Data Sheet
D0300G-10 Green Downward DAG in BacMam 10 ml Kit 687,00 PDF
U0300G-10 Green Upward DAG in BacMam 10 ml Kit 687,00 PDF
D0300R-10 Red Downward DAG in BacMam 10 ml Kit 687,00 PDF
U0300R-10 Red Upward DAG in BacMam 10 ml Kit 687,00 PDF
  • All prices are in EURO excl. VAT and shipping. Only available in Europe.

PIP2 Biosensor Assay Kits

Order# Description Amount Price Data Sheet
D0400G-10 Green PIP2 Sensor in BacMam 10 ml Kit 687,00 PDF
D0400R-10 Red PIP2 Sensor in BacMam 10 ml Kit 687,00 PDF
  • All prices are in EURO excl. VAT and shipping. Only available in Europe.

Red GECO Ca2+ Biosensor Assay Kits

Order# Description Amount Price Data Sheet
U0600R-10 Red GECO Ca2+ Sensor 10 ml Kit 687,00 PDF
U0600R-30 Red GECO Ca2+ Sensor 30 ml Kit 1379,00 PDF
U0600R-60 Red GECO Ca2+ Sensor 60 ml Kit 2071,00 PDF
  • All prices are in EURO excl. VAT and shipping. Only available in Europe.

ArcLight Voltage Biosensor Assay Kits

Order# Description Amount Price Data Sheet
D0700G-10 ArcLight Voltage Sensor 10 ml Kit 687,00 PDF
D0701G-10 Synapsin Promoter ArcLight Voltage Sensor 10 ml Kit 687,00 PDF
  • All prices are in EURO excl. VAT and shipping. Only available in Europe.

Download

  • Probes for Investigating Signal Transduction (Brochure) (PDF)

Literature

  • Cilia Have High cAMP Levels That Are Inhibited by Sonic Hedgehog Regulated Calcium Dynamics. PLoS ONE. 2016 Submitted.
  • Shigeto et al. (2015) GLP-1 stimulates insulin secretion by PKC-dependent TRPM4 and TRPM5 activation." J Clin Invest. 2015; 125(12):4714-4728. Pubmed
  • Arttamangkul et al. (2015) Does PKC activation increase the homologous desensitization of µ opioid receptors? Br J Pharmacy. 2015; 172(2):583-92. Pubmed
  • Chen et al. (2014) PKC-dependent Phosphorylation of the H1 Histamine Receptor Modulates TRPC6 Activity. Cells. 2014. 3(2): 247-257. Pubmed
  • Tewson et al. (2015) New DAG and cAMP Sensors Optimized for Live-Cell Assays in Automated Laboratories. J Biomol Screen. 2015. DOI: 10.1177/1087057115618608. Pubmed
  • Tewson et al. (2013) A multiplexed fluorescent assay for independent second-messenger systems: decoding GPCR activation in living cells. J Biomol Screen. 2013. 18, 797–806. Pubmed
  • Tewson et al. (2012) Simultaneous Detection of Ca2+ and Diacylglycerol Signaling in Living Cells. PLoS ONE. 2012. DOI: 10.1371/journal.pone.0042791. Pubmed
  • Kost et al. (2007) Implementation of BacMam virus gene delivery technology in a drug discovery setting. Drug Discov Today. 2007 May;12(9-10):396-403. Epub 2007 Mar 23. Pubmed