Plate reader, fluorescent assays

Fatty Acid Transporters. Figure 2 Quencher-based real-time fatty acid uptake assay with a fluorescently labeled FFA analogue (C1-Bodipy-C12). Predominantly protein-mediated fatty acid uptake by 3T3-L1 adipocytes (diamonds) was compared with diffusion-driven uptake by fibroblasts (squares) using the QBT Fatty Acid Uptake reagent (Molecular Devices Corp., CA, USA), which contains C1-Bodipy-C12 as substrate in conjunction with a cell impermeable quencher. Uptake kinetics was recorded using a Gemini fluorescence plate reader. Error bars indicate the standard deviations from 12 independent wells. RFU relative fluorescence units. 

Decisive for the success of the assay is the finding that the rate of oxidation constitutes a direct measure of the ee (Figure 8). High throughput was demonstrated by analyzing 100 samples in a 384-well format using a UV/fluorescence plate reader. Each sample contained 1 pmol of... 

Jones, P. B., Herl, L., Berezovska, O., Kumar, A. T. N., Bacskai, B. J. and Hyman, B. T. (2006). Time-domain fluorescent plate reader for cell based protein-protein interaction and protein conformation assays. J. Biomed. Opt. 11, 054024-10. 

Separation-based assays are preferred in many applications because they allow discrimination of signals due to substrate, product, and interference. When assays that involve fluorescence detection are developed, they are typically carried out by employing plate readers. When separation-based methods are employed for these applications, the influences of interferences (quenchers and other fluorescent compounds) on the final results are minimized because both substrate and product are quantified. With a separation-based approach, the label employed does not need to be placed in close proximity to the site of action of the enzyme, therefore minimizing the effect of the label on the mode of action of the enzyme. Of course, it is often desirable to develop assays that employ substrates free of labels. 

Gentest (now BD Biosciences) was the first to develop spectrophotometric assays to study CYP inhibition [98]. These assays are based on the turnover of mildly fluorescent substrate probes to moderately fluorescent metabolites, where metabolite formation is monitored by an increase in fluorescence using a plate reader [99,100]. Problems with these methods include background interference due to low signal-to-noise ratio, chemotype-specific interference and fluorescence quenching. Aurora Biosciences (now Vertex) has designed probes that exhibit larger fluorescence... 

All assays should be performed under reduced or F40 gold fluorescence lighting to minimize the potential for photodecomposition or activation. Assays are run in triplicate to determine percent inhibition. The tests are repeated at least once with a freshly prepared sample. If there is greater than 15% coefflciency of variation, the samples are run at least one additional time. When the reaction mixture is incubated within the plate reader, readings are taken immediately and at set times throughout the prescribed incubation period as established by the microsome supplier. For assays incubated outside of the plate reader, reactions were stopped in accordance with the product test procedure. 

Method B. Bisgaier et al. [78] reported a CETP assay using fluorescent cholesteryl 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-3-indacenedodecanoate (BODIPY-CE, Fig. 4) in microemulsions. Microemulsions for donor/acceptor contain triolein, BODIPY-CE/cholesteryl oleate, and l-hexadecanoyl-2-[cw-9-octadecenoyl]-s -glycero-3-phosphocholine. The assay mixtures consist of acceptor microemulsions, donor microemulsions, and a test sample in each well of flat-bottom 96-well plates. After preincubation at 37°C for 10 min, the reaction is initiated by addition of CETP solution. The fluorescent intensity in each well of the plates is periodically (every 10 sec) detected at 37°C with a fluorescent 96-well plate reader equipped with 485- and 538-nm bandpass filters in the excitation and emission paths, respectively. 

Some cellular assays also present the ability to measure kinase activation using recombinantly expressed intracellular sensor molecules that fluoresce in response to phosphorylation. These sensors are reviewed by Lawrence et al. (2007). The most interesting versions use yellow and blue fluorescent proteins to create a FRET through phosphorylation-induced folding with an intramolecular SH2 domain. SH2 domains are known to bind tightly to phosphotyrosine molecules and have been successfully shown to serve as phosphotyrosine detectors. In this type of assay, imaging is not necessary because the total fluorescence can be measured in a population of cells using a standard plate reader. 

An important consideration when determining volume is to ensure adequate space to accommodate the suspension of cells, the test compound, and one or more assay reagent. For assay protocols that use motion-induced mixing, the total volume may need to be lower to prevent loss of fluid and crosscontamination of wells. Changes in the surface-to-volume ratio of the culture medium may be important to consider when miniaturizing assays. For some fluorescence plate readers, the depth of medium may also be important and adjustments to the focal plane of the excitation light source may be necessary. 

In addition to the above plate readers, several other types are used to perform specific tasks in screening laboratories. Fluorescence kinetic plate readers can measure an assay plate quickly and... 

Subsequently, a 1 pL per well substrate solution is dispensed to the assay plates in another Multidrop-Combi equipped with plate stackers. After 30-min incubation at room temperature, the operator manually moves the assay plates to a plate detector with stackers and executes the measurement of assay plates in a specific detection mode (fluorescence intensity in this case). The data file from the plate reader and the tracer file will be copied to a computer for data analysis after the experiment. This screening platform is very useful for laboratories that screen small compound collections, assay validation with the LOPAC collection, and follow-up screens for hit confirmation and lead optimization. 

Excitation and emission wavelengths will depend on the fluorophore used, and should be optimized as much as is practical. The excitation and emission spectra of most common fluorophores are readily available (e.g., from Molecular Probes) if a variable-wavelength fluorimeter or fluorescence plate reader is used, then optimization is easy. If only fixed wavelengths are available, select those closest to the optima and verily that the fluorescence generated is sufficient for the intended assay. 

The vast majority of array assays have used fluorescence as the detection method due to the high sensitivity and the availability of detectors such as fluorescent plate readers for microtiter plates and high resolution DNA microarray scaimers for glass microarray slides. Therefore, the following discussion will focus on assay development coupled with fluorescent detection. 

---