- PHERAstar®FS multimode high-throughput microplate reader was used to access Gq coupled GPCRs
- HitHunter® is a robust and sensitive homogeneous ﬂuorescence polarization assay to measure IP3 levels
- Assay miniaturization up to 1536-well plate format
Table of contents
G protein coupled receptor (GPCR) activation regulates cell signaling via several second messengers, including 3’-5’-cyclic AMP (cAMP), inositol phospholipids and calcium. The quantitation of accumulation of these second messengers is used to pharmacologically characterize both the action of GPCR ligands and to identify novel compounds in high-throughput screening (HTS). GPCRs coupling to Gαs and Gαi proteins activate or inhibit, respectively, adenylate cyclase, subsequently changing intracellular cAMP levels. GPCRs coupling to Gαq or Gαo proteins activates phosphoinositol phospholipase C-ß, an enzyme that hydrolyzes phosphatidylinositol-4,5- bisphosphate (PIP2) to form sn-1,2-diacylglycerol and inositol- 1,4,5-trisphosphate (IP3). IP3 binds and opens endoplasmic IP3 gated calcium channel, resulting in the release of bound calcium into the cytosol. Metabolic products of IP3 also modulate cell signaling, such as inositol 1,3,4,5-tetrakis-phosphate (Ins P4), which synergistically facilitates IP3, mediated calcium release. DiscoverX has developed a homogeneous assay based on ﬂuorescence polarization (FP), to measure IP3 levels generated by GPCR activation. This application note, describes the use of IP3 assay along with PHERAstar FS multi-mode HTS plate reader.
The assay is based on FP mode using a proprietary binding protein that is highly selective for the active isomer of IP3. The assay is a competitive binding assay (ﬁgure 1), in which cellular IP3 displaces a ﬂuorescent derivative of IP3 from a speciﬁc binding protein. The assay measures changes in FP, a single wavelength ratiometric technique, in which a ﬂuorescent derivative of IP3 is used as a tracer. In the assay unlabelled IP3, either a standard IP3 solution, or derived from the cell lysate, displaces the tracer from the binding protein and the rotation time increases and low FP signal is measured. By this means, a calibration is generated to the standard IP3 dilutions and the molar concentration of IP3 in the cell lysate is determined by interpolation.
Materials & Methods
BMG LABTECH’s PHERAstar FS combines rapid plate reading necessary for HTS with the enhanced performance and sensitivity needed to read small ﬂuid volumes. The multi-mode microplate reader has the ﬂexibility to excel with the most demanding assays and is designed to read all leading HTS detection modes (ﬂuorescence intensity, time-resolved ﬂuorescence, ﬂuorescence polarization, luminescence and absorbance) in all formats up to 1536. The PHERAstar FS was run in FP mode for the monitoring of the HitHunter® IP3 demo kit (DiscoverX Corp.) containing the following reagents:
|HitHunter IP3 kit reagents|
|1||IP3 Standard – 20 μM|
|2||IP3 Standard Dilution Buffer|
|3||Perchloric Acid (PCA) – 0.2 N|
|4||IP3 Tracer - Green|
|5||IP3 Binding Protein|
The standard curve for DiscoverX’s IP3 kit was run according to the package insert protocol in black 384-well plates (non-binding polypropylene plates; Greiner), and the ﬂuorescence polarization signal was read on the PHERAstar FS one hour after the addition of the last reagent using the following protocol for plate reader setup (ﬁgure 2).
The table below outlines the volumes and procedures for the IP3 protocol measuring both standard curve and agonist/antagonist treated cells in a 384-well plate format. Measuring agonist/antagonist treated cells, IP3 levels were stabilized with the addition of 5 μL of 0.2 N perchloric acid.
Table 1: HitHunter® IP3 protocol for standard curve and agonist/antagonist treated cells.
|HitHunter IP3 Protocol|
|Condition||Standard Curve||Cells Agonist||Cells Antagonist|
|Step 1: Standard/ cells||10 μL standard||10 μL cells||10 μL cells|
|Step 2: Antagonist||-||-||5 μL antagonist|
|Incubate||-||-||30 min. at 37° C|
|Step 3: Agonist||5 μL water||5 μL agonist||5 μL agonist|
|Incubate||-||20 seconds (room temperature)|
|Step 4: PCA||5 μL PCA -0.2N|
|Step 5: Tracer||10 μL IP3 Tracer-Green|
|Step 6: Binding Protein||20 μL IP3 Binding Protein|
|Gently tap plates for even mixing (shake plates for 5 minutes)|
|Read fluorescence polarization signal on the PHERAstar FS |
(Excitation filter 485 nm/Emission filter 520 nm)
Results & Discussion
DiscoverX Hithunter® IP3 was prepared in 384-well format and standard curves were run on BMG LABTECH’s PHERAstar FS in ﬂuorescence polarization mode. IP3 standard reagents were added according to the assay protocol and the ﬂuorescence polarization signal was read one hour after the addition of the last reagent. The Hithunter® IP3 standard curve is illustrated in the ﬁgure below (ﬁgure 3).
The Hithunter® IP3 assay is a robust, sensitive and speciﬁc tool for measuring cellular D-myo-inositol 1,4,5-triphosphate. The signal is based on competitive binding between an IP3 ﬂuorescent tracer and an unlabeled IP3 from cell lysate or IP3 standard. The signal is read as a change in ﬂuorescence polarization and is inversely proportional to the amount of IP3 in cell lysates. The standard curve signal to background is >5, and the EC50 is 13 nM. The signal can be measured immediately or up to 16 hours later.
GPCRs are critical targets in HTS drug discovery and GPCR signaling can be examined by direct quantitation of IP3 by applying the HitHunter® IP3 kit. Good results were obtained on the PHERAstar FS multi-mode microplate reader which is designed to read all leading HTS detection modes in formats up to 1536. The high degree of sensitivity, easy-to-use software, robust hardware and optimized detection systems make the PHERAstar FS ideal for GPCR analyses in the high-throughput assay environment.
The PHERAstar was run in FP mode for the HitHunter® IP3 assay, which uses FP for sensitivite detection and ease of use. The assay is ideal for the detection of basal levels of IP3 in cell lysates, as well as low levels for IP3 induction. Once the cells are lysed, the stable assay signal can be read the same day or overnight. There are few robust IP3 assays currently available and none easily scalable to 1536 automation. DiscoverX offers this assay to give maximum precision and reliability for GPCR screening.
For more information on DiscoverX assays please refer to the website: www.discoverx.com
HitHunter, DiscoverX and the DiscoverX logo are registered trademarks of the DiscoverX Corporation.