SpectraMax M2 and M2e Microplate Reader User Guide
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0112-0102 E
The Cisbio Bioassays HTRF
®
(Homogeneous Time-Resolved Fluorescence) technology is a
proprietary Time-Resolved Fluorescence technology that overcomes many of the drawbacks
of standard Fluorescence Resonance Energy Transfer (FRET) techniques, such as the
requirements to correct for autofluorescence and the fluorescent contributions of unbound
fluorophores.
Optimizing Time-Resolved Fluorescence Read Mode
The SpectraMax M2 enables you to read Time-Resolved Fluorescence assays from the top of
a plate. The SpectraMax M2e enables you to read Time-Resolved Fluorescence assays from
either the top or the bottom of a plate. You should use solid white plates for top Time-
Resolved Fluorescence reads, and white plates with clear bottoms for bottom reads.
Two important settings to obtain the best results in TRF assays are integration delay and
integration time.
Integration delay: The amount of time that elapses between the flash of the lamp
(excitation) and the beginning of data acquisition from the well.
Integration time: The amount of time the well is read.
Delay and integration time are usually specified in the package insert of commercially
available TRF reagent kits. If you do not use a kit, then start with a delay of 50 µs and try
different delays up to 400 µs with a fixed integration time of 400 µs. After you select the
optimum delay (based on the highest ratio of a well that contains a fluorophore divided by
wells that contain only buffer) optimize the integration time, which is typically between 400
µs and 1000 µs.
If the Time-Resolved Fluorescence assay you use has low signal or gives results with high %
CV, then use 100 reads per well. If a faster read speed is required, then in the Settings dialog,
turn the Settling Time to Off and experiment with fewer flashes per well until you achieve an
acceptable precision and speed.
Read Types
The instrument support the following read types:
Endpoint
In an endpoint read type, a reading of each plate well is taken in the center of each well, at a
single wavelength or at multiple wavelengths. Raw data values are reported as optical
density (OD), % transmittance (%T), or relative fluorescence units (RFU).
Kinetic
In a kinetic read type, the instrument collects data over time with multiple reads taken in the
center of each well at regular intervals. To achieve the shortest possible interval for kinetic
reads, choose wavelengths in ascending order.
The software can do the following calculations based on raw data: VMax, VMax per Sec, Time
to VMax, and Onset Time. Kinetic readings can be single wavelength or multiple wavelength
readings.
