Automated Plate Handling for Walk-Away Functionality
Molecular Devices is advancing cellular and array based assays by providing instrumentation that images the entire well, cell by cell, at true high throughput screening rates. The ImageXpress® Velos System is a laser scanning microplate imaging system with proprietary collection optics for high content cell-based assays, colony morphology studies, and multiplexed spot and bead arrays. Assays can be run in plate densities from 6-1536 wells. Here we report on the implementation of the Autorun software feature for “walk-away” plate processing with the ImageXpress Velos System laser scanner integrated with the Twister II Microplate Handler (Caliper Life Sciences, Hopkinton, MA).
The Twister II Microplate Handler integration provides automation to the ImageXpress Velos System. The base configuration, with one three-rack pod, provides plate capacity of up to 80 microplates and can be expanded with additional pods to handle up to 320 plates. The handler arm provides five axes of motion with an arm extension of 8.5” and wrist rotation of 270° to accommodate any source/destination configuration. Scanning and loading/unloading speeds allow total cycle times of 3-4 minutes per plate at 10 micron sampling regardless of well density. The ImageXpress Velos System-HTS has a small footprint which fits on standard lab benches (Figure 1).
The Autorun user interface is flexible and easy to use. The ImageXpress Velos System-HTS can be optionally configured with a barcode reader for positive plate identification, tracking, and file naming. Room temperature incubation and plate scanning delays can be incorporated into your assay methods. The system can run with or without plate lids and up to four separate ImageXpress Velos System Methods per plate can be run with timed repeats for kinetics over minutes or hours. For example, for the Mitotic Count Assay (Application Note No. 8), the single ImageXpress Velos System Method defines all the image acquisition, image analysis, and assay criteria required for automatic mitotic cell quantification by two-color image processing.
Autorun: Powerful Features and Easy to Use Interface
Using ImageXpress Velos System with a Twister II Microplate Handler enables the user to choose Autorun to set up the ImageXpress Velos System for automated plate handling. The Setup/ Run menu option allows simple set up (Figure2).
- Upon opening the window for Setup/Run, the available stations appear in green. “Nest” refers to the ImageXpress Velos System instrument (plate nest), the barcode reader station, if present for scanning a microplate is referred to as “Barcode”, and the de-lidding area is called “Lid”. The number of configured racks on Twister II will also be green.
- No. of cycles allows each plate to be scanned up to 41 times.If a number greater than 1 is specified, you must choose the amount of time to delay between each scan. Individual delays between scans may be entered between 1 and 4,096 minutes (68 hours).
- Selecting Abort on Error will stop the Autorun program if a fatal error occurs during the run. The Error will be recorded into an Autorun log file of the run and all plate activity will halt until a user intervenes to restart the program.
- If a barcode reader is detected on the system, enabling Use Barcodes results in each plate being scanned by the barcode reader before being placed into the ImageXpress Velos System plate tray.
Setup Rack Options
- No. of Plates is selected for the number of plates to be automatically run or the box Do All can be checked and all plates in the rack will be scanned and delivered to the destination rack (Figure 3).
- Restack can be enabled to return the plates to the source rack in their original order after scanning on ImageXpress Velos System. One of the Racks must always be left empty to receive finished plates before restacking. If Restack is checked, after all the plates in the rack that have been configured to run are read, and stacked in the destination rack, the plates will subsequently be restacked in the source rack
- Remove Lids will be active if a de-lidding station is detected. By checking Remove Lids, plates will be uncovered before loading into ImageXpress Velos System.
- Method file(s) allows you to browse and as shown in Figure 3 up to four Methods can be used for scanning, analyzing, and saving data from each plate
Autorun Advanced Settings
In the Autorun pull down menu, the Advanced option allows the ImageXpress Velos System-HTS to be used in a “simulated” robot mode to enable multiple readings of the same plate without intervention by the user (Figure 4). This feature is useful for scanning a plate at different time intervals when running kinetic studies or for reading a plate with up to four different Methods. The setup and run of an automated time series of reads on a plate, or multiple Methods on a plate, is identical to the setup with the actual use of the plate handling robot.
Results and Discussion
To demonstrate Autorun, paclitaxel treated HeLa cells in a 96-well plate were fixed, permeabilized and immunostained with a mitotic marker antibody directed against the phosphorylation of Ser28 of histone H3 (Active Motif; Carlsbad, CA). The primary antibody against phospho histone H3 was detected with a secondary antibody labeled with Alexa Fluor-488 (AF488; Invitrogen;Carlsbad, CA) in the plate top half showing rows A-D selected (the selected wells show blue outline in Figure 3). In rows E-H the secondary antibody was labeled with Chromeon 488 (Ch488; Active Motif).
Figure 3 shows the use of four ImageXpress Velos System Methods with Autorun. The wells selected for Method MI AF488- 10 and Method MIAF488-5 for two-color image acquisition from rows A-D (selected wells shown with blue outline in Figure 3) of a 96-well plate at 10 x 10 or 5 x 5 micron sampling, respectively. The Method MI Ch488-10 and Method MI Chr488-5 acquires images from rows E-H of a 96-well plate at 10 x 10 or 5 x 5 micron sampling, respectively.
Image acquisition and automatic mitotic index (MI) cell quantification by two-color image processing has been described previously in Application Note No. 8 “High-Throughput Two-Color Mitotic Index Assay”. Whole well images were analyzed on a cell-by-cell basis by identifying cells in the red (PI stained) channel and using the background corrected integrated fluorescence intensity for the green (AF488 or Ch488) and red channels. With Autorun the well images, a list file (.csv) enumerating cell-by-cell data for each well and a summary text file showing the percent mitotic cells in a plate layout format are automatically saved.
ImageXpress Velos System-HTS offers a complete automated yet cost-efficient benchtop system with integrated image acquisition and analysis with the flexibility important for automatic multiple plate, multiple scan, and multiple Methods for walk-away processing. The ImageXpress Velos System- HTS provides a time saving alternative to custom plate loaders and a money saving alternative to larger, more complex integrated systems. It only takes a moment to set up an entirely unattended run.
The ImageXpress Velos System-HTS is an attractive solution to those laboratories in need of a user friendly automated plate handling platform with integrated image acquisition and analysis modules with the flexibility important for developing, running, and validating image analysis procedures under high throughput conditions. This platform enables simple “plug and play” applications to meet the needs of life science research in both academia and industry. BBI invites you to contact us to discuss your specific application. We offer full customer support in assay development, validation, and automation.