Premo™ Halide Sensor
Chloride channels are involved in a variety of important physiological functions that include transepithelial ion transport, cellular electrical excitability and ion homeostasis. Defects in chloride channel function underlying a number of chronic disease states and they therefore represent valuable drug targets. To facilitate measurements of chloride channel activity Invitrogen has developed Premo™ Halide Sensor, a fluorescent protein based biosensor.
The Premo™ Halide Sensor is a pharmacologically relevant sensor for functional studies of ligand- and voltage-gated chloride channels. It is based on a halide-sensitive form of Venus, the brightest available YFP molecule, affording an excellent signal window. Packaged in BacMam format, the transient expression is efficient in a range of cell types, including human primary cells. The combination of BacMam delivery and the bright fluorescence of Venus provide a highly sensitive, robust and easy-to-use tool for efficient screening of halide ion channels and transporter modulators in a range of mammalian cellular models. The spectral properties of the Premo™ Halide Sensor are compatible with all standard HTS platforms.
- Chloride channel–specific—functional assay for voltage- and ligand-gated chloride channels and transporters
- Fast—measure chloride flux in high-throughput mode with highly reproducible results
- Robust—reliably high expression, no-wash format and excellent signal window
- Convenient—study chloride channel activity in your cellular model by efficient and non-cytopathic delivery of Premo™ Halide Sensor by BacMam technology
- Pharmacologically relevant—known modulators show dose-dependent quenching and BacMam delivery enables assays in primary cells
Premo™ Halide Sensor Assay
Figure 2. Principle of the Premo™ Halide Sensor.
To uncouple cell maintenance and preparation from screening, simply freeze transduced cells and bring out as little as 4 hours before the screen. Both stable cell lines and human primary cells can be prepared frozen and “assay-ready”.
Principle of the Premo™ Halide Sensor
The combination of genetically encoded biosensors with BacMam delivery technology yields robust and easy-to-use cell-based assays. BacMam (baculoviral) particles carrying the Venus gene (yellow) are taken up by endocytosis. The DNA traffics to the nucleus where only the CMV promoter-driven Venus gene is transcribed; baculovirus promoters are not recognized by the mammalian transcriptional machinery—hence no virus replication and no toxicity. Following transcription, the Venus mRNA is expressed in the cytosol and cells are ready to assay. This process begins within 4-6 hours after transduction and in many cell types is completed after an overnight period. In the absence of halide ions, the YFP halide sensor fluoresces brightly; on influx of halide ions (particularly the iodide surrogate ion) fluorescence is quenched (Figure 2).
Premo™ Halide Sensor Workflow
1. Add virus to cells for 2 to 4 hours.
2. Treat with an enhancer for 2 hours.
3. Incubate overnight and plate or freeze for future use.
In particular U-2 OS cells are efficiently transduced (near 100%) and are recommended for initial assay development work. However, BacMam has been shown to efficiently transduce multiple mammalian cell types, including human primary and stem cells. Transduced cells can be divided into aliquots for a homogenous cell population and stored frozen. Assay-ready frozen cells can be plated as little as 4 hours prior to the assay.