cAMP HiRange detection using the SpectraMax Multi-Purpose Plate Reader
Introduction
In this application note, we show how to use the SpectraMax® i3, SpectraMax® Paradigm® and SpectraMax® M5e multi-function microplate readers for reliable, high-throughput HTRF® testing with a perfect Z' Factor and high repeatability EC50 values.
HTRF® is a versatile technology developed by CisbioBioassays to detect biomolecular interactions1. This technology combines fluorescence resonance energy transfer (FRET) technology with time-resolved (TR) fluorescence to eliminate short-lived background fluorescence. This assay uses donor and acceptor fluorophores. When the donor and acceptor are close enough to each other, an energy source (such as a xenon flash lamp) excites the donor to transfer energy to the acceptor, which emits fluorescence of a particular wavelength.
HTRF uses four specialized fluorophores that can be combined into a pair of donor-acceptor TR-FRET groups. The donor is a cryptate of Eu (+3) and a cryptate of lanthanum ( Lumi4TM-Tb), and its long-lived fluorescence can be used in time-resolved fluorescence detection. The two receptors are XL665 and d2, respectively, for HTRF detection. The excitation spectra of both receptors overlap with the emission spectra of the HTRF donor. The emission peak of each acceptor is at 665 nm and the donor does not emit light or emit light in this range. The original HTRF receptor XL665 is a phycobiliprotein pigment purified from red algae. The second generation receptor d2 is a modified allophycocyanin that is 100 times smaller than XL665 and can reduce the steric hindrance that may occur in the XL665 assay.
The HTRF cAMP HiRange kit is capable of quantifying cyclic adenosine monophosphate (cAMP, cyclic adenosine 3', 5' monophosphate) in a cell sample. cAMP is a key second messenger in G protein coupled receptor (GPCR) signaling. When the ligand binds to the GPCR, a change in concept occurs, and activation of the receptor then activates the G protein. Further signal transduction depends on the type of activated G protein. Activation of Gs causes adenylate cyclase to regulate upregulation of cAMP. The free cAMP produced in the cell competes with the d2-labeled cAMP for binding to the anti-cAMP cryptate antibody. Therefore, an increase in intracellular cAMP leads to a decrease in FRET and a decrease in the detected 665 nm fluorescence emission (Fig. 1).
Advantage
- Highly reliable homogeneous experiment
- Z' factor ≥ 0.9
- Process and stable high throughput (HTS) testing
- SoftMax Pro software preset experiment template for quick results
material
- cAMP HiRange 1000 test
(Cisbio P/N 62AM6PEB)
- Black and white low volume 384-well microplate
(Greiner P/N 784076 and 784075)
- SpectraMax i3 multi-function microplate reader
(Molecular Devices)
- SpectraMax Paradigm Multi-Purpose Plate Reader (Molecular Devices)
- SpectraMax M5e Multi-Purpose Microplate Reader
(Molecular Devices)
- HTRF test cartridge
(Molecular Devices P/N 0200-7011)
method
The HTRF cAMP HiRange kit is supplied by CisbioBioassays. CAMP standards with final concentrations ranging from 0.17 nM to 2800 nM were prepared according to the cAMP HiRange HTRF drug insert. Positive control (maximum FRET) without cAMP and negative control without cAMP or cAMP-d2 are included. 20 μL of reagent was added to each well as described in Table 1.
data analysis
The HTRF detection was analyzed using a Cisbio patented ratio algorithm based on dual emission wavelength detection. The donor at the 616 nm emission wavelength serves as an internal reference, while the acceptor at the 665 nm emission wavelength serves as an indicator for determining the biological response. This ratiometric measurement reduces the difference between pores and pores and reduces compound interference. The Delta F calculated in step 4 reflects the comparison of the signal at the time of the measurement relative to the background size for internal measurements. The results are calculated from the 665 nm/616 nm ratio and are expressed as Delta F as follows:
The Z' factor was calculated from negative (no cAMP, no cAMP-d2) and positive control (no cAMP).
Generate and analyze data using SoftMax® Pro software, including some preset HTRF experiment templates to simplify inspection and analysis.
result
Data analysis and mapping were performed as described above using a 4-parameter curve fit of SoftMax Pro software. The best results were obtained according to the plate reader parameter settings of Table 2. When the delay time, integration time, and number of pulses increase, a decrease in DF% is observed. The use of a black low volume 384-well microplate can increase the detection window from 3020 to 3253 compared to a white microplate (Figure 2). However, the Z' factor, the detection window and standard deviation and the EC 50 values ​​considering positive and negative control were very similar in the two well plate types (Table 3). (Note: When using the SpectraMax M5e Multi-Purpose Plate Reader, a white plate is required.)
The SpectraMax i3 and SpectraMax Paradigm readers obtained the same results when comparing the same test plates one by one (Figure 3). For this test, a white microplate was utilized. The detection windows are 3004 and 3051, and the Z' factors are 0.92 and 0.90, respectively. The EC50 value was 19.1 nM compared to the published value (Table 3). The SpectraMax M5e Multi-Purpose Plate Reader gives similar Z' factor and EC50 values.
The SpectraMax Paradigm reader benefits from the dual PMT configuration and is faster than other readers, allowing it to read two HTRF emission wavelengths simultaneously (Table 3).
to sum up
The SpectraMaxi3 and SpectraMaxParadigm readers are equipped with HTRF-certified test cartridges with high throughput screening capabilities. When used on the SpectraMax Paradigm reader, this cartridge is capable of dual simultaneous detection to speed up readings. In the three plate readers, the perfect Z' factor measured by cAMPHiRange indicates the reliability of the multi-function detection system. The HTRF preset experimental template included with SoftMaxPro software greatly simplifies data acquisition and analysis.
reference
1. http://
2. Zhang, JH, Chung, TDY, and Oldenburg, KR (1999). A simple statistical parameter for use in evaluation and validation of high throughput put screening assays J. Biomolecular Screening 4(2): 67-73.
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