Dr. Paul Held, the Laboratory manager at BioTek Instruments, is an early tester and adopter of Persomics Arrays.
Dr. Held joined BioTek in 1993 as a research scientist and is currently the Application Laboratory Manager. His responsibilities include managing the day to day operations of the laboratory as well as developing applications on BioTek instrumentation. He holds a BS in Pharmacy, an MS in Pathology and Molecular Biology and a Ph.D. in Molecular Biology.Prior to joining BioTek, Dr. Held was a postdoctoral research fellow at the University of Vermont.
BioTek entered the automated imaging market with the launch of the Cytation 3 Cell Imaging Multi-Mode Reader comprised of an automated digital microscope combined with BioTek’s patented Hybrid Technology plate reader optics. Recently it was followed by the the Cytation 5, adding two additional microscopy modes, laser-based autofocus capabilities and software features enhancing the use of Persomics Arrays.
Persomics approached BioTek to test gene silencing arrays on the Cytation platforms and to validate the complete end-user workflow for the Persomics ImagineArraysTM on their exciting new imagers.
Dr. Held had early success validating Persomics’ prototype Validation ImagineArrays, which have multiple replicates of benchmark control genes. The validation arrays are ideal for optimization of cell line culture conditions, seeding density and staining protocols prior to using library based arrays. As shown in figures 1 through 3 below, Dr. Held quickly generated striking images using the Cytation 3 and 5 imaging systems; demonstrating the utility of this technology for bench top functional genomics and RNAi screening. He also validated cost effective methods for automating the entire fixation and staining of cells on Persomics arrays, which will be discussed in detail at the end of this article.
Figure 1. Example images of a Persomics ImagineArray (top) and an enlarged sub-section of the array (bottom). Montage images are created by stitching multiple images together to represent a larger field of view on the array (this array was roughly 7mm x 15mm). Each spot silences a single gene with siRNAi and is demarcated with a red fluorescent dye.
Figure 2. Three color overlay images of Control, RELA and INCENP RNAi Microspots acquired on the Cytation Imaging System. Overlaid images show the expected phenotypes in HeLa cells due to silencing of benchmark control genes RELA/p65 and INCENP.
Panel A: control cells showing no change in single nuclei (blue) and immunolabled (green) RELA/p65 protein over the red microspots or compared to surrounding cells.
Panel B: Expected phenotype showing loss of RELA/p65 protein only in cells over the microspot.
Panel C: shows the multinucleated phenotype from INCENP silencing on the microspot.
Figure 3. RELA phenotype with the red Dye removed. Notice the extent of RELA/p65 silencing. Virtually no green stain appears over the location of the spot.
Workflow Automation for Staining Persomics Arrays with BioTek MultiFlo FX
It is possible to make your life even easier by automating the entire staining procedure for Persomics Arrays prior to imaging. This could be set up for just a couple or a stack of Persomics plates. The entire process becomes semi-automated using a BioTek MultiFlo FX. An ImageArrayTM plate can contain up to 3200 spots; as such it acts as the equivalent of thirty 96-well microplates, saving considerable amounts of reagents and requiring considerably less hands on time to run at the bench. The processing of a single ImagineArray plate for imaging takes approximately 2 hours from the addition of paraformaldehyde fixative through cellular staining protocols. During the two hours there is little hands on time, as most of the process is devoted to 1° and 2° antibody binding incubations rather than hands-on interaction with the plate. This is the ideal scenario for automation when staining multiple plates and using an automated stacker.
The BioTek MultiFlo FX was used to dispense the required reagents, as well as provide and aspirate PBS wash solution. Incubation timing was controlled by LHC software. With a plate stacker a large number of plates can be processed in parallel in several hours.
Figure 4. Schematic of the ImagineArray Plate Processing Steps. The MultiFlo FX was used to dispense the required reagents, as well as provide and aspirate PBS wash solution. Incubation timing was controlled by LHC software (BioTek Instruments). Following fixation and staining regions of the ImagineArrayTM plate were imaged with a 20X objective using a montage of images to assess multiple microspots in their entirety.
Dr. Held recently published both a poster and an application note about use of the Persomics Arrays with Cytation imaging systems. They are available to download here: High Content RNA1 Screening Using Printed Libraries
Application Note, soon to come
We look forward to Dr. Held’s participation in testing the first Persomics full human kinome ImagineArrays that are currently in development at Persomics. Thanks Paul!