Predetermined Residual Volumes of Assay Buffer in High Content Screening Assays

High Content Screening (HCS) assays by and large involve the stimulation of cellular events followed by the fixation, staining, and permeabilization of cells to allow for the introduction of antibodies to pinpoint and quantify myriad cellular processes at the protein level. This workflow requires multiple cell wash steps as illustrated in this example of a gluocokinase translocation assay recently published.

Figure 1: Workflow for Gluocokinase Translocation Assay. Green text indicates assay component addition to microplate; red text indicates operations performed by microplate washer; black text shows incubation conditions; and blue text informs on imaging reader used: GE Healthcare’s IN Cell 3000 Analyzer.

In this publication, the ELx406 microplate washer was used to wash the cells and leave behind specific residual volumes of assay buffer at the last dispense/aspiration step of the wash cycle. The principle lever associated with accurate provision of residual volumes in the ELx406 is the aspiration manifold z-height setting that can be controlled using either the on-board keypad or Liquid Handling Control software. Many factors can affect accurate residual volume precision beyond the obvious aspiration manifold z-height and microplate well volume. These include well shape, microplate composition (polystyrene, polypropylene, uncoated, coated, etc.) and assay buffer composition (ionic strength, detergents, non-specific binding additives, etc.) In this blog, we present some guidelines for achieving residual volumes without a significant amount of trial and error.

It is recommended that Corning tissue culture-treated plates be used for accurate provision of residual volumes without significant influence from assay buffer additives, especially for lower well-densities such as 96-well microplates. The table provided below may serve as a guideline for achieving residual volumes in tissue culture-treated plates based on aspiration manifold z-height settings.



Note: settings may vary dependent on different manufacturers microplates and assay buffer not tested in this study.

Additional information regarding residual volumes using BioTek plate washers can be found in the Tech Note.

What types of assays are you currently performing that require residual volume to be left after the final aspirate? What buffers and settings are you currently using?

Fast, Full Plate Magnetic Bead Washing for Multiplexed Assays

BioTek’s industry standard ELx405 Microplate Washer is now available for use with multiplexed assays using magnetic beads. The ELx405 Magnetic Bead Washer has been developed in conjunction with leaders in the provision of genotyping, focused gene expression and protein assays utilizing Luminex MagPlex microsperes.

ELx405 is a 96-/384-well microplate washer which incorporates a neodymium iron boron magnet for immobilizing magnetic beads on the bottom of the microplate during wash steps providing high bead recoveries and washing efficiency. The 96-tube manifold (also available in 192-tube for more rapid washing in 384-well densities) provides rapid full-plate processing of multiple washes and wash cycles typical of Luminex xMAP-based assays.

The use of the ELx405 Magnetic Bead Washer provides ease-of-use, improved precision in replicate data and the ability to automate the Luminex xMAP technology for multiple applications such as the quantification of human metabolic hormones from sera or plasma. Below is the 13-plex calibration curves from a Milliplex panel run using ELx405.


Precision in the replicate data provided by the ELx405 was compared to that provided using standard filter-based xMAP sample preparation in the histogram below.

Higher precision is evident with ELx405 with a significantly greater number of replicates with %CV ≤ 5 and there are no high %CV replicates as those seen with filter-based sample preparation which are indicative of filter clogging.

Do you view the benefits of ease of use, higher precision and automation-capability provided by MagPlex beads and ELx405 as useful improvements to Luminex xMAP technology? Will your lab adopt the new MagPlex magnetic microspheres or are you content with the original polystyrene microspheres and filter-based sample preparation?

Welcome to "Scientifically Speaking"

After years of isolation and experimentation we've coaxed our busy scientists out of the lab.... at least for a bit. Our scientific team has joined forces to author BioTek's first blog. You'll see postings from multiple authors as they share expertise and thoughts about their work. Their goal is to provide the life science research field and microplate instrumentation users with cutting edge, informative, helpful, and (sometimes) entertaining tips, tricks, articles, tech resources, videos and much more.

Please join our ongoing discussions by leaving comments. If you have questions or thoughts on topics, let us know. We're looking forward to connecting with you!

BioTek Instruments, Inc., headquartered in Winooski, VT, USA, is a worldwide leader in the design, manufacture, and sale of microplate instrumentation and software. BioTek instrumentation is used to facilitate the drug discovery process, to advance discoveries in genomics and proteomics, and to aid in the advancement of life science research.