Fluorescent Protein-Based Biosensors

  • Genetically encoded calcium sensor for iterative assays
  • Ratiometric, FRET-based detection for quantitative results
  • Highly efficient delivery system for large-scale cell transfection and batch freezing
  • Easy-to-use and efficient delivery system

Life Technologies introduces the new Molecular Probes® Premo™ product line, combining the stability of genetically encoded ion indicators and environmental sensors with the efficiency of BacMam delivery1. The first in a series of products, the Premo™ Cameleon Calcium Sensor is a no-wash, non-organic dye indicator for intracellular calcium signal measurements. Based on the YC3.60 version of a GFP-based sensor family developed by Tsien, Miyawaki, and co-workers,2,3 the binding of four Ca2+ ions to a calmodulin-M13 moiety induces a conformational change (Figure 1). This change brings the cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) domains closer, allowing fluorescence resonance energy transfer (FRET) to occur.

The ratiometric aspect of the readout–an increase in YFP emission (535 nm, green-yellow emission) and a decrease in CFP emission (485 nm, blue emission)–significantly reduces assay variations due to compound or cellular autofluorescence, non-uniform cell plating, differences in expression levels between cells, instability of instrument illumination, and changes in illumination pathlength.

The Premo™ Cameleon Calcium Sensor is provided as a ready-to-use baculovirus stock containing the Cameleon DNA, which is efficiently delivered to target cells prior to cell plating. The stock is used in conjunction with a Premo™ enhancer for increased sensor expression in multiple cell types, including primary and stem cells (Table 1). The Premo™ Cameleon Calcium Sensor readily and accurately detects intracellular calcium flux from different receptors. Standard pharmacological assays for multiple GPCR agonists and antagonists have been tested. An example of the robustness, reproducibility, and accuracy of the system is demonstrated using the endogenous histamine receptor using histamine, pyrilamine, and thioperamide in HeLa cells (Figure 2).The Premo™ Cameleon Calcium Sensor provides a very sensitive and accurate method for live cell imaging analysis that can be used to test multiple compounds in the same sample. An example of the temporal response of Premo™ Cameleon Calcium Sensor–transduced cells stimulated with ATP is illustrated in Figure 3 (image and video loop). Increases in Ca2+ were detected almost immediately and by 32 seconds after the addition of the ATP, Ca2+ levels had returned to normal.

The no-wash, no-dye format and ratiometric readout eliminates wash steps that can dislodge cells, reduces data variability, and increases data integrity. Withthis unique format, other potential errors associated with washing procedures are eliminated as well. Expression levels will be maintained for several days, enabling iterative assays to be run; for instance, when examining agonist/antagonist relationships on the same cells. The combination of the Premo™ Cameleon Calcium Sensor and the efficient, ready-to-use BacMAM technology in our first Premo™ product offers a unique combination of highly selective intracellular Ca2+ measurement with a very efficient and ready-to-use viral transduction delivery system.

References 1. Kost, T. et al. (2005) Nature Biotech. 23:567. 2. Miyawaki, A. et al. (1997) Nature 38:882. 3. Nagai, T. et al. (2004) PNAS 101:10554.

Schematic of premo
Figure 1. Schematic of Premo™ Cameleon Calcium Sensor mechanism. The eCFP and cpVenus units of cameleon are connected by a calcium-sensitive moiety consisting of a calmodulin and a M13 domain. Upon binding calcium the calmodulin-M13 moiety undergoes a conformational change that brings the fluorescent protein-domains into close proximity. The resulting FRET signal allows ratiometric measurements of free intracellular calcium, independent of well-to-well (or cell-to-cell) differences in gene expression levels.
Histamine receptor
Figure 2. Measurement of endogenous histamine receptor calcium response in HeLa cells. A. HeLa cells transfected with the Premo™ Cameleon Calcium Sensor were treated with histamine to determine the EC50 concentration. B. A separate plate was used to evaluate an antagonist dose response with pyrilamine and thioperamide in the presence of an EC50 concentration of histamine. Calculations were performed to determine the Z′ values at specific points on the dose response curves. Pyrilamine is a known H1 receptor antagonist that couples through Gq proteins and the second messenger Ca2+. Thioperamide is a known H3 receptor antagonist that couples through Gi proteins and the second messenger cAMP.


Live cell of calcium flux
Figure 3. Live cell measurement of calcium flux. Porcine left atrial appendage progenitor cells were transfected with Premo™ Cameleon Calcium Sensor. The following day ATP was applied to the cells at a final concentration of 20 µM and imaging performed using a Zeiss 5 Live high speed confocal system (Carl Zeiss MicroImaging, Inc., Thornwood NY). Excitation source was a 405 nm diode laser (50 mw) operated at 50% power. Emission was collected simultaneously on two linear CCD detectors using a 490 nm dichroic mirror to split the beam through a 415–480 nm bandpass filter for CFP and a 550 nm longpass filter for YFP. Images were collected at a rate of 10 frames per second (512 x 512 pixels) using a 40x Plan-Neofluar 1.3 NA oil immersion objective lens. Data provided from collaboration with Dr. Kenton Gregory, Oregon Medical Laser Center Bioimaging Suite, Providence St. Vincent Hospital, Portland, Oregon.

Cell lines and cell types transduced with Premo™ Cameleon Calcium Sensor

Cell line

Agonist

CHO M1WT3Carbachol - EC50 ~ 23 nM
CHO K1Carbachol- no response
COS7Thrombin - EC50 ~ 10 units/ml
HEK 293ATP/carbachol - EC50~ 9.1 µM
HeLaHistamine- EC50 ~ 260 nM
HematopoieticNot currently amenable
HepG2ATP- EC50 ~ 6.5 µM
Left atrial appendage progenitorcells-adult porcine stem cellsATP
NIH 3T3Transduction only
Nt2 & ep2002 hEC cellsTransduction only
Primary cardiac muscle cellsCarbachol
Primary skeletal muscle cellsATP
Rat primary astrocytesTransduction only
Rat primary hippocampal neuronsTransduction only
SKBR3ATP- EC50 ~ 2.8 µM
U2OSATP- No receptor/no response