Research. Image FlashPlate

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1 Drug Discovery Research Image FlashPlate Clinical Screening Application Note Use of Image FlashPlate on the Wallac ViewLux ultrahts microplate imager The Wallac ViewLux is an ultra high throughput microplate imager for all major fluorescence, absorbance, and luminescence technologies. The detector is a back illuminated CCD camera operating at -100 Celsius coupled to an optimized telecentric optical lens. The camera and lens systems operating in the luminescence detection mode are suitable for radiometric assays. Image FlashPlate is a scintillant coated 384-well small volume microplate designed for non-separation high throughput screening assays using a variety of isotopes. The emission maximum of the scintillant is >600 nm (red-shifted) in order to allow the plate to be imaged on the ViewLux. The plate wells are conical-shaped, which allows optimal image capture by the camera. The plate has a standard microplate footprint that is compatible with current plate and liquid handling equipment. It is particularly well suited for binding assays that utilize a coated microplate well to capture a target molecule. Because the ViewLux reads entire plates in one exposure, throughput is not effected by plate density. Imaging times for radiometric Image FlashPlate assays are typically 5 minutes or less per plate. The ViewLux Workstation software is compatible with Windows NT. It allows full control over image acquisition and image display. Results are automatically stored in the ViewLux database. Also the data or images can automatically be exported for analysis by other applications. The instrument supports both robot loading and batch mode operation. Up to 64 plates can be loaded for unattended operation. Users can alternate between batch and robot loading according to needs.

2 CONVERSION GUIDELINES To convert a 96-well microplate assay or a 384-standard well to a 384-shallow well assay, it is important that both the coating and the assay conditions be optimized for the miniaturized format. The Conversion Guidelines below list some recommended starting conditions and considerations for adapting a 96-well or 384- standard well assay to the Image FlashPlate 384-shallow well format. I. Coating Plates Starting conditions for transferring a coating process from a 96-well or 384-standard well format to the Image FlashPlate 384-shallow well format follow: The same concentrations of protein used to coat a 96-well or 384-standard well microplate or a bead is likely to be near the optimum in many cases. It is always best to titrate the concentration of coating protein to determine the optimal coating concentration. Start with the same plate coating protocol (washes, blocking solutions, etc.) that is currently being used. Coating and blocking volumes in the Image FlashPlate 384-shallow well plates are best in the range of 20 to 25 µl per well. Greater than 25 µl per well may cause plates to stick together if they are stacked during the coating process. II. Assay Protocol A starting point for evaluating assay conditions for the Image FlashPlate assay is to use the same reagent concentrations, incubation times, etc. as the protocol for a 96-well or 384-standard well assay. However: Optimal concentrations of some reagents may NOT be the same as for the equivalent 96-well or 384-standard well assay. Sensitivity or absolute counts may be improved by adding the same mass of tracer and/or standard/test compounds as used in the 96-well or 384-standard well assay, but in a smaller, more concentrated volume. Reagent volumes typically are 1/10 the volume used in a 96-well microplate and less than 1/2 the volume used in 384- standard well microplate. The total assay volume in the well should be kept to 20 µl or less. The order of addition of reagents should also be examined when miniaturizing a microplate assay. Small volumes used in the Image FlashPlate wells (e.g. 5 µl) may not flow to the bottom of the well due to surface tension. Adjusting the reagent volumes such that the last addition is the largest volume ( 10 µl) can help wash all the additions to the bottom of the well. Avoid sealing the plate (TopSeal, PerkinElmer Life Sciences, Catalog #SMP201) until you are ready to count the Image FlashPlate. Removing the seal between assay steps can cause the contents to be drawn into adjacent wells. Use a rigid lid or another plate to cover the assay between steps to reduce evaporation if there is any delay. All 384-well microplates with a square well design from all manufacturers have a tendency for sample to wick up the corners of the wells due to capillary action. The Image FlashPlate has rounded wells, which minimizes this effect. However, assays at elevated temperatures (37 C), extended incubations (more than 24 to 48 hours), assays with large volumes per well (more than about 20 µl per well) as well as other conditions may cause some amount of wicking. If your assay must include these conditions you should verify that wicking is not causing the contamination of adjacent wells. 2

3 PRECISION CHARACTERISTICS The precision of the Image FlashPlate was tested by adding a 20 µl aqueous solution of [ 125 I]BSA to each well, followed by incubation of the covered plate for 18 hour at room temperature. Plates were imaged on the ViewLux (4x4 binning) for 5 minutes. The individual intra-plate CVs were plotted (Figure 1) and found to average 7.4%. % CV ViewLux Signal Figure 1. Signal and %CV performance for two separate lots of Image FlashPlate measured on the ViewLux. DMSO TOLERENCE Image FlashPlate has been shown to be tolerant of up to 2 µl of 100% DMSO spotted directly on the plate and stored for 2 hours at room temperature, or for 2 weeks at -20 C prior to assay. The assay consisted of adding a 20 µl solution of [ 3 H] biotin in ethanol to the wells of the plate, allowing the plate to dry overnight, and then measuring the signal on the ViewLux. % CONTROL Pre-spot Volume 0.5µL 1.0µL 2.0µL H 2 O Control % DMSO % DMSO Figure 2. Effect of DMSO on Image FlashPlate tritium counting efficiency measured on the ViewLux. STREPTAVIDIN COATING Image FlashPlates were coated with streptavidin using the same protocol as used for the basic Streptavidin FlashPlate -HTS (Perkin Elmer Life Sciences, Catalog #SMP410). The below table compares the key parameters between the basic FlashPlate and the Image FlashPlate streptavidin coating. To determine the specificity of biotin binding to streptavidin coated wells, [ 3 H] biotin (PerkinElmer Life Sciences, Catalog # NET721) was premixed with cold biotin (Sigma, Catalog # B4501) at varying concentrations. 20 µl of pre-mixed reagent was added to each well. The Image FlashPlate was then incubated overnight at room temperature. Signal was obtained by reading plate with a ViewLux with 4x4 binning for 5 minutes, as shown in Figure 3. Parameter FlashPlate-HTS Image FlashPlate (384-Standard Well Format) (384-Shallow Well Format) Coating volumes Streptavidin 50 µl 20 µl Blocking and wash buffers 70 µl 25 µl Coating conditions Coating conditions (concentrations, buffer formulations, wash and blocking conditions) were identical for both the 384 well formats. Assay volumes 50 µl 20 µl Incubation times and conditions Other assay conditions were identical for the two formats Performance Curve position (B 50% ) 3 pmol/well 4 pmol/well Between well (within plate) CV% <10% <10% Capacity ( 3 H Biotin) 15.5 pmol /well 6.94 pmol/well 3

4 ViewLux Signal % B/B0 Figure 3. Displacement of [ 3 H] biotin by cold biotin on Image FlashPlate. ADENYLYL CYCLASE ACTIVATION ASSAY Cyclic AMP (camp) plays a crucial role in inter-cellular communication by functioning as a second messenger. The binding of a hormone or neurotransmitter to its receptor can either stimulate or inhibit the rate of formation of camp. This is accomplished by modulating the enzymatic activity of adenylyl cyclase, a membrane associated enzyme that catalyzes the conversion of ATP to camp. The standard Adenylyl Cyclase Activation FlashPlate Assay (Perkin Elmer Life Sciences, Catalog #SMP710) allows direct measurement of receptor mediated adenylyl cyclase activation/inhibition in G proteincoupled receptor systems. The protocol is a fully homogeneous cellular assay, which measures a true second messenger response. Image FlashPlate was coated with anticamp antibody in order to generate a camp standard curve. Reagents from the Adenylyl Cyclase Activation Assay (SMP710) were used in this assay. 5µL of camp standard was added to each well followed by 5µL of stimulation buffer. The plate was incubated at room temperature for 45 minutes followed by addition of 10 µl of detection buffer containing [ 125 I] camp. The plate was incubated at room temperature for 18 hours and read on the ViewLux (4x4 binning for 5 minutes). The results are shown in Figure 4. Figure 4. Standard curve of camp competition binding measured on the ViewLux. WHEAT GERM AGGLUTININ COATING Wheat Germ Agglutinin (WGA) is a dimeric protein with a molecular weight of 36 kda. WGA is a plant lectin that can bind carbohydrate residues on glycosylated proteins. The simplest sugar structure recognized by WGA is N-acetylglucosamine (NAG). WGA coated FlashPlates can be used to capture G protein-coupled membrane preparations via their associated carbohydrates. Image FlashPlates were coated with WGA using the same protocol as used for the basic WGA-coated FlashPlates (Perkin Elmer Life Sciences, Catalog #SMP411). The specificity of binding is demonstrated by a competition binding assay using unlabeled glycophorin to displace [ 125 I] glycophorin as shown in figure 5. Glycophorin is a glycosylated blood protein known to bind to WGA. Signal Figure 5. Displacement of [ 125 I] glycophorin by cold glycophorin on Image FlashPlate. 4

5 PROTEIN TYROSINE KINASE ASSAY An assay for the kinase activity of the protein tyrosine kinase Src was developed on Image FlashPlate. Streptavidin coated Image FlashPlates were incubated with a buffered solution of the biotinylated tyrosine kinase peptide substrate (PKS1) (Boehringer Mannheim, Catalog # ) and [ 33 P] ATP (Perkin Elmer Life Sciences, Catalog #NEG302H). A serial dilution of Src Kinase (Upstate Biotechnology, Catalog #14-177) was added and the plate incubated at 37 C for three hours. The wells were aspirated and then washed once with 0.1% Triton X- 100 diluted with 1XPBS and five times with 1XPBS. The plate was read on the ViewLux (4x4 binning) for 5 minutes to give the results shown in Figure 6. The activity of Src Kinase shown above can be inhibited by the kinase inhibitor staurosporin. One unit per well of Src kinase was inhibited by a dilution series of staurosponin under the same assay conditions as described above. The plate was incubated for 3 hours at 37 C, aspirated, washed and read on the ViewLux (4x4) binning to give the inhibition curve shown in Figure 7. Signal Figure 7. Inhibition of Src kinase activity by staurosporin. Signal PRODUCTS AVAILABLE Plates supplied by PerkinElmer Life Sciences Figure 6. Src Kinase activity demonstrated on Image FlashPlate. Basic Image FlashPlate plates RP100E Basic Image FlashPlate plates RP100 FlashPlate is a registered trademark of Packard BioScience Company under U.S. patent #5,496,502 and foreign equivalents. 5

6 Worldwide Headquarters: PerkinElmer Life Sciences, 549 Albany Street, Boston, MA USA (800) European Headquarters: PerkinElmer Life Sciences, Imperiastraat 8, BE-1930 Zaventem Belgium Technical Support: in Europe: techsupport.europe@perkinelmer.com in US and Rest of World: techsupport@perkinelmer.com Belgium: Tel: France: Tel: Netherlands: Tel: Germany: Tel: United Kingdom: Tel: Switzerland: Tel: Italy: Tel: Sweden: Tel: Norway: Tel: Denmark: Tel: Spain: Tel: MicroBeta is a registered trademark and VICTOR, ViewLux, Wallac and PerkinElmer are trademarks of PerkinElmer Life Sciences, Inc , Jan 02 Printed in Finland by Offset House Oy Naantali 2002