Caroline Cardonnel | Sr. Applications Scientist | Molecular Devices
Introduction
HTRF® is a versatile technology developed by Cisbio Bioassays for detecting biomolecular interactions1. It combines fluorescence resonance energy transfer (FRET) technology with time-resolved (TR) measurement of fluorescence, allowing elimination of short-lived background fluorescence. The assay uses donor, either europium cryptate (Eu3+) or terbium (Lumi4™-Tb) cryptate, and acceptor fluorophore, either XL665 or the small molecule d2. When donor and acceptor are close enough to each other, excitation of the donor by an energy source (e.g., flash lamp) triggers an energy transfer to the acceptor, which in turn emits specific fluorescence at a given wavelength.
Here, we show how SpectraMax® i3x and SpectraMax® iD5 Multi-Mode Microplate Readers are used to perform Gq-coupled receptor assays using HTRF technology. G-protein coupled receptors signal through two major pathways, regulation of cAMP and increase in intracellular calcium levels mediated by IP3. Cisbio’s IP-One Gq kit offers an alternative to calcium flux assays by enabling detection of the accumulation of inositol monophosphate (IP1), a stable downstream metabolite of IP3 (Figure 1).
Figure 1. Activation of the Gq pathway. IP1, a downstream metabolite of the second messenger IP3, accumulates in the presence of LiCl
In the presence of lithium chloride (LiCl), the degradation of IP1 is inhibited and accumulates in signaling cells (Figure 1). In the IP-One HTRF assay, IP1 labeled with d2 acceptor competes with native IP1 produced by the cells for binding to an IP1-specific monoclonal antibody labeled with cryptate donor. An increase in production of unlabeled IP1 by the cells results in disruption of TR-FRET and a decrease in the HTRF signal (Figure 2)2. The IP-One assay can be used to characterize compounds that act on Gq-coupled receptors in either adherent or suspension cells.
Figure 2. Principle of the HTRF IP-One competitive binding assay. IP1 labeled with d2 acceptor competes with native (unlabeled) IP1 for binding to the donor-labeled IP1-specific monoclonal antibody. An increase in unlabeled IP1 results in disruption of TR-FRET and a decrease in the HTRF signal
Materials
- IP-One Gq kit, 1000 tests (Cisbio cat. #62IPAPEB
- iCell® Neurons (Cellular Dynamics International)
- SpectraMax i3x Multi-Mode Microplate Reader (Molecular Devices cat. #I3X)
- HTRF Detection Cartridge (Molecular Devices cat. #0200-7011)
- SpectraMax iD5 Multi-Mode Microplate Reader (Molecular Devices cat. #ID5)
- HTRF Detection System (Molecular Devices cat. #6590-0144; includes Enhanced TRF Module, excitation and emission filters)
Methods
IP1 standards with final concentrations ranging from 1.9 nM to 7700 nM were prepared as indicated in the assay product insert3. A positive control without any unlabeled IP1 (maximum FRET) and a negative control without IP1 or anti-IP1-d2 antibody were included. Reagents were dispensed to a final volume of 20 μL per well as indicated in Figure 3. The plate was incubated for one hour at room temperature and read with the SpectraMax i3x and iD5 readers using the optimized settings shown in Table 1.
| Standard | Serial dilutions | IP1 working solution nM | IP1 final concentration nM |
|---|---|---|---|
| Std7 | 50 µL IP1 Std reconstitued + 950 µL StimB | 11000 | 7700 |
| Std6 | 50 µL Std7 + 150 µL StimB | 2750 | 1925 |
| Std5 | 50 µL Std6 + 150 µL StimB | 688 | 481.6 |
| Std4 | 50 µL Std5 + 150 µL StimB | 172 | 120.4 |
| Std3 | 50 µL Std4 + 150 µL StimB | 43 | 30.1 |
| Std2 | 50 µL Std3 + 150 µL StimB | 11 | 7.7 |
| Std1 | 50 µL Std2 + 150 µL StimB | 2.7 | 1.9 |
| Std0 | 150 µL StimB | 0 | 0 |
*StimB has to be first diluted with distilled water from 5X to a 1X solution (e.g. 1 volume of StimB 5X + 4 volumes of distilled water)
Figure 3. Assay setup for a 384-well low-volume plate. ‘StimB’ is Stimulation Buffer, included in the IP-One Gq kit.
| SpectraMax i3x | SpectraMax iD5 | |
|---|---|---|
| Tb donor/red acceptor | Tb donor/red acceptor | |
| Required components | HTRF Detection Cartridge | HTRF Detection System |
| Excitation | 340 nm | 340/70 nm |
| Emission | Donor: 620 nm Acceptor: 665nm |
Donor: 616/10 nm Acceptor: 665/10 nm |
| Number of flashes | 30 | 30 |
| Integration delay | 30 µs | 20 µs |
| Integration time | 400 µs | 200 µs |
| Other | Run Microplate Optimization and Read Height Adjustment when running the assay for the first time, as well as when using a new lot of plates or a different assay volume (Settings > More Settings > Show Pre-Read Optimization Options in SoftMax Pro Software). | |
Table 1. Instrument settings for SpectraMax i3x and iD5 readers. These settings were optimized for the IP-One Gq assay and may differ slightly from settings used with other HTRF assays.
To optimize the settings, the delay time, integration time, and the number of pulses were adjusted until the Delta F% (see Data Analysis, below) increased noticeably. The optimized settings listed in Table 1 can be transferred to any plate reader of the same model, i.e. settings listed below for the SpectraMax iD5 reader can be used for any SpectraMax iD5 reader. However, microplate optimization and read height adjustment should be performed any time a new plate type—or lot of plates—or reagent volume is used so that the optics for the reader are aligned to the assay plate. This ensures minimal error, as well as optimal assay sensitivity and dynamic range.
Data analysis
Analysis of HTRF assays uses Cisbio’s patented ratiometric reduction method based on the two emission wavelengths detected. Donor emission at 616 nm is used as an internal reference, while acceptor emission at 665 nm is used as an indicator of the biological reaction being assayed. This ratiometric measurement reduces well to-well variation and eliminates compound interference. Delta F, calculated in step 4 below, reflects signal to background of the assay and is useful for inter-assay comparisons. Results are calculated from the 665 nm/616 nm ratio and expressed in Delta F as follows:
Data were generated and analyzed using SoftMax® Pro Software, which contains preconfigured HTRF protocols to simplify detection and analysis using the method described above.
Results
The standard curves for SpectraMax i3x and iD5 readers were graphed using SoftMax Pro Software and applying a 4-parameter curve fit (Figure 4), with the results summarized in Table 2. The assay suitability was assessed using the EC50, which should be below 150 nM. For both readers, the EC50 values were below 85 nM, well within the acceptable range. Cisbio defines an acceptable signal to noise ratio as ≥ 20, and both readers met this criterion. With these results, as well as CV values below 6% for all standards, both the SpectraMax i3x and the SpectraMax iD5 readers demonstrate high sensitivity and a wide dynamic range for the IP-One assay.
Figure 4. HTRF IP standard curve. HTRF IP1 standard curve measured on the SpectraMax i3x (green circles) and SpectraMax iD5 (red circles) readers. The assay window for the SpectraMax iD5 was somewhat larger but the assay quality was excellent for both readers (CV < 6%).
| Parameter | Passing | SpectraMaxi3x | SpectraMaxiD5 |
|---|---|---|---|
| EC50 | ≤ 150nM | 63.1 nM | 84.1 nM |
| Signal/noise | ≥ 20 | 22.4 | 24.5 |
Table 2. Results summary for HTRF IP1 standard curve.
Conclusion
The SpectraMax i3x and iD5 readers can be equipped with an HTRF-certified detection cartridge or an HTRF-certified detection system, respectively. Both readers demonstrated their ability to detect IP1 concentrations according to Cisbio’s acceptance criteria. Data acquisition and analysis are simplified using SoftMax Pro Software with preconfigured HTRF protocols.
References
- http://www.htrf.com/htrf-technology
- Garbison KE, Heinz BA, Lajiness ME. IP-3/IP-1 Assays. 2012 May 1. In: Sittampalam GS, Grossman A, Brimacombe K, et al., editors. Assay Guidance Manual.
- https://www.cisbio.eu/media/asset/c/i/cisbio_dd_pi_62ipapeb-62ipapec-62ipapej.pdf