Chapter 5: Read Modes and Read Types
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Concentrations or qualitative results are derived from raw data with a standard curve or by
comparison with reference controls.
Applications of Luminescence
Chemiluminescent or bioluminescent reactions can be induced to measure the quantity of a
particular compound in a sample. Examples of luminescent assays include the following:
Reporter gene assays (the measurement of luciferase gene expression)
Quantitation of adenosine triphosphate (ATP) as an indication of cell counts with cell-
proliferation, cytotoxicity, and biomass assays
Enzyme measurements with luminescent substrates, such as immunoassays
Optimizing Luminescence Assays
Luminescence can be read from the top or the bottom of a plate. You should use solid white
plates or white plates with clear bottoms for luminescence reads.
For standard luminescence a separate light path without monochromators carries the emitted
light to a dedicated PMT. The optimal emission wavelength is between 360 nm and 630 nm.
To read only one luminescent event in the well, you can get the best sensitivity using the
standard luminescence measurement, without a wavelength selected.
Luminescence read times are not designated by multiple reads per well, but rather by the total
integration time you enter (between 1 ms and 1500 ms). Typical luminescence assays require
between 500 ms and 1000 ms integration.
If wells are incubated for a long period of time, you should mix the plate before the read. This
can be done using the Shake setting.
If it seems that the signal is always higher in the first wells read (for example, column A), you
should dark adapt the plate to reduce the auto-luminescence of the white plastic. To help
eliminate background luminescence from a plate that has been exposed to light, you should
dark adapt the plate by placing the sample-loaded plate inside the instrument for several
minutes before you start the read.
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), relative fluorescence units (RFU), or relative light units (RLU).
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.
The kinetic read type can collect data points in time intervals of seconds, minutes, or hours (up
to 99 hours).
