Application Note

Optimized HTRF settings for SpectraMax multi-mode microplate readers

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Caroline Cardonnel | Sr. Applications Scientist | Molecular Devices

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

HTRF-compatible SpectraMax

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:

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

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