Screening
imagers and automated plate preparation plat- forms can be introduced. To do so, HTS are con- ducted mainly with end point assays using fixed cells allowing the decoupling of the sample prepa- ration from image acquisition, as well as the shar- ing of the plate preparation systems between vari- ous projects.
Figure 2
Example of a 1536-well plate heat map displayed in the data analysis software. By clicking a well, the respective image is displayed
A typical process for a high-throughput HCI screen in 1536-well plates could be the following: First, cells are cultivated and plated into 1536- well assay plates using the automated cell culture platform SelecT (TAP Biosystems). Then these assay plates are transferred on to a plate prepara- tion platform (Agilent) to perform compound transfer with an Echo 550 (Labcyte) as well as reagent dispensing, incubation, fixation and washing. Once the cells are fixed, the complex and non-homogenous immuno-staining protocols are performed on a dedicated automated platform using the Catalyst 5 robot (Thermo Scientific) and designed especially for this purpose. The sys- tem is equipped with high density washer/dis- penser BNX1536 (Bionex) as well as Cytomat incubators (Thermo Scientific) necessary for incu- bation at various temperatures and illumination conditions (Figure 1). The immuno-staining plat- form in NIBR was implemented to gain addition- al flexibility by decoupling the compound addi-
tion and fixation steps from the antibody staining process. Once plate preparation is completed, the plates are stored at 4˚C until they can be meas- ured on one of the imagers available in the screen- ing unit.
High-content imagers and image analysis For an imager, the following features are important to perform high-throughput assays: ability to han- dle 1536-well plates and high-speed image acquisi- tion (20-100 mins per 1536-well plate). Further- more it is advantageous to perform image analysis in parallel to image acquisition (ie ‘on the fly’ analysis). Finally, the storage capacity of the instru- ment should be high enough to cope with terabytes of data, or alternatively be set up for automated data transfer to a dedicated database. The choice of the imager is dependent on two assay require- ments: resolution and throughput. For assays requiring sub-cellular resolution and high through- put, our preferred imager is the Opera QEHS (PerkinElmer), a confocal imager equipped with four lasers and four CCD cameras allowing on-the- fly image analysis. For assays requiring high throughput based on fluorescence intensity meas- urements with no need for sub-cellular resolution, the Acumen eX3 (TTP LabTech), a plate scanning device equipped with three lasers allowing on-the- fly analysis of the fluorescence intensity distribu- tion, is optimal. For medium-throughput HCI we generally utilise the IN Cell Analyzer 2000 (GE) which is a wide-field imager equipped with a large- chip CCD camera. It can handle 96, 384 and 1536- well plates; however with image analysis being decoupled from the image acquisition process. Performing the follow-up assays for primary hits identified with a laser scanning device such as Acumen by high resolution images from Opera or InCell200 can improve throughput tremendously. These three instruments complement each other well and the choice of the optimal imager as well as the image analysis software are key criteria to exploit at best the full potential of the technology for HTS.
A high-content imager usually creates a specific image format linked to metadata (eg channel, objective lens, pixel resolution etc) which makes the use of third party software more difficult since it might require an adaptation/conversion of the image or file format. This can slow down the screening process and increase the image storage space making the use of third party analysis soft- ware cumbersome. For HTS campaigns, the sim- plest solution is to use the image analysis software provided with the imager since no transfer of
22 Drug Discovery World Winter 2011/12
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