FlexStation 3 Benchtop Multi-Mode Microplate Reader User Guide
110 0112-0127 B
Optimizing Time-Resolved Fluorescence Assays
Time-resolved fluorescence (TRF) assays can be read from the top or
bottom of a microplate. Solid white plates are recommended for top
time-resolved fluorescence reads, and white plates with clear bottoms
are recommended for bottom reads.
If the time-resolved fluorescence assay you are using has low signal or
gives results with high % CV, use 100 readings per well. If a faster read
speed is required, be sure Settling Time is Off in the SoftMax Pro Plate
Settings dialog, and experiment with fewer flashes per well until
acceptable precision and speed are achieved.
Important settings for obtaining the best results in TRF assays are
integration delay and integration time:
• The integration delay is the amount of time that elapses
between the flash of the lamp (excitation) and the beginning of
data acquisition from the well (emission).
• The integration time is 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 a kit is not used, start with a
delay of 50 μs and try different delays up to 400 μs with a fixed
integration time of 400 μs. After the optimum delay is chosen, based on
the highest ratio of a well containing a fluorophore divided by wells
containing only buffer, optimize the integration time, which is usually
between 400 μs and 1000 μs.
FlexStation_manual.book Page 110 Tuesday, June 25, 2013 10:22 AM
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