Monday, June 29, 2009

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.
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.


Guidelines 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?

By: BioTek Instruments

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