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.
Montana Molecular Assay Steps Diagram
- 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
- 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
- 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).
- Multiplex Calcium and DAG
- DAG Assay on a Plate Reader
PIP2 Sensor – PIP2 is a red, dimerization-dependent fluorescent protein sensor under the control of a CMV promoter. When PIP2 is hydrolyzed,
red fluorescence decreases. This sensor can be multiplexed with the green sensors for cAMP or DAG. When co-expressed with a green dimerization-dependent monomer, GA, this sensor becomes a fluorescent
protein exchange (FPX) sensor that increases in green fluorescence as the red fluorescence decreases.
- Green PIP2 Sensor Data
Red GECO Ca2+ Sensor – The red-fluorescent, genetically encoded Ca2+ indicator for optical imaging (R-GECO) developed by Robert Campbell’s lab at
University of Alberta. R-GECO can be paired with cADDis, the green-fluorescent cAMP sensor, or the green-fluorescent Upward and Downward DAG sensors to simultaneously detect both Ca2+
and cyclic AMP or Ca2+ and diacylglycerol in living cells. The baculovirus delivery system enables consistent expression in a wide variety of cell types.
- Multiplex Calcium and DAG
ArcLight Voltage Sensor – A genetically encoded, fluorescent voltage sensing protein in a BacMam viral delivery system is a noninvasive, easy-to-use tool for studying the electrical activity
of live neurons.
- Visualizing Electrical Activity in Cells Using a GFP-based Voltage Probe ArcLight (Video 1)
- Visualizing Electrical Activity in Cells Using a GFP-based Voltage Probe ArcLight (Video 2)