Introduction
HTRF assays from Cisbio have evolved to encompass the areas of G protein-coupled receptors, kinases and cell signaling, epigenetics, and biomarkers. A wide variety of toolbox reagents and kits are available to address protein-protein interactions, nuclear receptor assays, receptor dimerization, ligand binding, receptor internalization, enzyme assays, and many more.
HTRF involves the use of two fluorophores, a donor and an acceptor, to label proteins or other biomolecules of interest. When the biomolecules interact, their proximity enables fluorescence resonance energy transfer (FRET) to occur from the donor to the acceptor fluorophore. Using long-lifetime fluorescent lanthanide cryptates as donors allows time-resolved detection of HTRF assays, minimizing short-lived background fluorescence from compounds and other materials. The detection of time-resolved FRET (TR-FRET) with a microplate reader provides a readout of biomolecular binding.
Getting the best HTRF results requires a microplate reader that has been validated to meet the HTRF performance specifications set by Cisbio. A reader’s optics, energy source, and detectors, as well as the settings used to detect HTRF, all impact performance. Our readers use optimized components for HTRF detection, including an HTRF detection cartridge for the SpectraMax i3x and Paradigm Multi-Mode Microplate Readers, and an enhanced TRF module plus validated filters for the SpectraMax iD5 reader.
Cisbio’s HTRF certification program guarantees that readers bearing the “HTRF compatible” logo meet the specifications required for optimal HTRF performance. This technical note presents the optimized settings validated by Cisbio for Molecular Devices HTRF-compatible microplate readers.
Molecular Devices HTRF-compatible microplate readers
- SpectraMax® i3x Multi-Mode Microplate Reader
- HTRF Detection Cartridge (Molecular Devices P/N 0200-7011, or custom TR-FRET for Tb/green)
- SpectraMax® iD5 Multi-Mode Microplate Reader
- HTRF Detection System (Molecular Devices P/N 6590-0144; includes Enhanced TRF Module, excitation and emission filters)
- SpectraMax® Paradigm® Multi-Mode Microplate Reader
- HTRF Detection Cartridge (Molecular Devices P/N 0200-7011, or custom TR-FRET for Tb/green)
- SpectraMax® M5e Multi-Mode Microplate Reader
- FlexStation® 3 Multi-Mode Microplate Reader
Figure 1. HTRF-compatible SpectraMax Multi-Mode Readers. SpectraMax i3/i3x and Paradigm readers can be equipped with the HTRF detection cartridge. The SpectraMax iD5 reader uses the HTRF detection system, which includes an Enhanced TRF Module and filters for excitation and emission.
About the reader setup recommendations
HTRF assays are available in three different donor/acceptor fluorophore combinations, each requiring specific reader settings for optimal results:
- Europium cryptate donor / red acceptor (Eu/red)
- Terbium cryptate donor / red acceptor (Tb/red)
- Terbium cryptate donor / green acceptor (Tb/green), Tag-lite
SpectraMax i3/i3x readers
| Parameters | Settings | |
|---|---|---|
| Europium cryptate donor / red acceptor, Terbium cryptate donor / red acceptor | ||
| Optical configuration | Cartridge | HTRF |
| Read mode | TR-FRET | |
| Read type | Endpoint | |
| Wavelengths | Fixed per cartridge | Ex 340, Em1 616, Em2 665 |
| Plate type | User selectable | White plate required for high-performance HTRF measurement |
| Read area | User selectable | |
| PMT and optics | Number of pulses Excitation time (fixed) Measurement delay Integration time Read height |
30 0.05 (fixed) 0.03 ms 0.4 ms Plate format- and volume-dependent; optimize for each new plate type or volume used |
| Terbium cryptate donor / green acceptor, Tag-lite | ||
| Optical configuration | Cartridge | Custom TR-FRET (Tag-lite HTRF) |
| Read mode | TR-FRET | |
| Read type | Endpoint | |
| Wavelengths | Fixed per cartridge | Ex 340/80, Em1 616/15, Em2 665/10 |
| Plate type | User selectable | White plate required for high-performance HTRF measurement |
| Read area | User selectable | |
| PMT and optics | Number of pulses Excitation time (fixed) Measurement delay Integration time Read height |
30 0.05 (fixed) 0.03 ms 0.4 ms Plate format- and volume-dependent; optimize for each new plate type or volume used |
SpectraMax Paradigm reader
| Parameters | Settings | |
|---|---|---|
| Europium cryptate donor / red acceptor, Terbium cryptate donor / red acceptor | ||
| Cartridge | HTRF | |
| Read mode | TR-FRET | |
| Read type | Endpoint | |
| Wavelengths | Fixed per cartridge | Ex 340, Em1 616, Em2 665 |
| Plate type | User selectable | |
| Read area | User selectable | |
| PMT and optics | Number of pulses Excitation time (fixed) Measurement delay Integration time Read height |
30 0.05 (fixed) 0.03 ms 0.5 ms Plate format- and volume-dependent; optimize for each new plate type or volume used |
| Terbium cryptate donor / green acceptor, Tag-lite | ||
| Cartridge | Custom TR-FRET (Tag-lite HTRF) | |
| Read mode | TR-FRET | |
| Read type | Endpoint | |
| Wavelengths | Fixed per cartridge | Ex 340/80, Em1 616/15, Em2 665/10 |
| Plate type | User selectable | |
| Read area | User selectable | |
| PMT and optics | Number of pulses Excitation time (fixed) Measurement delay Integration time Read height |
30 0.05 (fixed) 0.03 ms 0.5 ms Plate format- and volume-dependent; optimize for each new plate type or volume used |
SpectraMax iD5 reader
| Parameters | Settings | |
|---|---|---|
| Europium cryptate donor / red acceptor, Terbium cryptate donor / red acceptor | ||
| Read mode | TR-FRET | Enhanced TRF module must be installed |
| Read type | Endpoint | |
| Wavelengths | Use filters | Ex 340/70 Em1 665/10 Em2 616/10 |
| Plate type | User selectable | |
| Read area | User selectable | |
| PMT and optics | Flashes per read Excitation time (fixed) Measurement delay Integration time Read height |
50 0.05 (fixed) 0.1 ms 0.6 ms Plate format- and volume-dependent; optimize for each new plate type or volume used |
SpectraMax M5e and FlexStation 3 readers
| Parameters | Settings | |
|---|---|---|
| Europium cryptate donor / red acceptor | ||
| Read mode | TR-FRET | |
| Read type | Endpoint | |
| Wavelengths | Number of wavelength pairs: 2 | Ex 314; Em1 620, with 570 cutoff; Em2 665, with 630 cutoff |
| Plate type | User selectable | White plate required for high performance HTRF measurement |
| Read area | User selectable | |
| TRF settings | Integration delay Integration time |
50 300 |
| PMT and optics | Flashes per read | 50 to 100 |
| Terbium cryptate donor / red acceptor | ||
| Read mode | TR-FRET | |
| Read type | Endpoint | |
| Wavelengths | Number of wavelength pairs: 2 | Ex 340; Em1 620, with 570 cutoff; Em2 665, with 630 cutoff |
| Plate type | User selectable | White plate required for high performance HTRF measurement |
| Read area | User selectable | |
| TRF settings | Integration delay Integration time |
50 300 |
| PMT and optics | Flashes per read | 50 to 100 |
| Terbium cryptate donor / Green acceptor, Tag-lite | ||
| Read mode | TR-FRET | |
| Read type | Endpoint | |
| Wavelengths | Number of wavelength pairs: 2 | Ex 340; Em1 520, with 495 cutoff; Em2 620, with 570 cutoff |
| Plate type | User selectable | White plate required for high performance HTRF measurement |
| Read area | User selectable | |
| TRF settings | Integration delay Integration time |
50 300 |
| PMT and optics | Flashes per read | 50 to 100 |
Conclusion
SpectraMax multi-mode microplate readers with time-resolved fluorescence detection capability were fully tested for HTRF performance by Cisbio using their standard certification methods and exhibited excellent performance while exceeding the requirement criteria for HTRF certification. Optimized detection cartridges or filters are available to support the full range of HTRF assays including Tag-lite