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The edge effect in microplate assays

microplate lids

Microplate assays are widely used for many biochemical and cell-based assays. Maintaining a consistent environment in all wells of a microplate is essential for generating reliable and accurate data. However, variabilities are often observed in the data obtained from wells at the edges of a plate compared to wells located toward the plate center – a phenomenon known as the edge effect. An edge effect can occur in both cell-based and biochemical assays and in all microplate formats (96 wells, 384 wells, and 1536 wells)

What causes the edge effect

The edge effect is caused by variabilities in the environment in different wells of a microplate, which may be attributed to various factors. Notably, the evaporation rate may differ among wells located at the edges and towards the center of a plate. This, in turn, may lead to changes in reagent concentration or the pH of wells at the edges and in the center of a plate, which may have many effects on cells, including cell metabolism or attachment. Even though it can be observed in all microplate formats, the effect of evaporation is more pronounced in plates with a higher number of wells as they have lower sample volume per well. In addition, thermal gradients across a microplate may cause an edge effect in temperature-sensitive assays. 

How the edge effect influences assay results

The edge effect can lead to both higher and lower values in the wells at the edge of a plate than in the wells towards the plate center. However, in both scenarios the edge effect can cause greater standard deviations, which negatively affect data reliability. In addition, in some instances, the edge effect can lead to assay failure or increased cytotoxicity.

Strategies to reduce or eliminate the edge effect

Some labs routinely leave the outside rows of microplates empty to prevent the edge effect. However, this approach reduces the number of wells available for experimental treatments. Therefore, various other strategies have been proposed to counteract the edge effect.

  • Reduction of the assay time, which is associated with lower evaporation and a less pronounced effect on the well environment.
  • Minimization of temperature gradients to reduce the edge effect in temperature-sensitive assays.
  • Use of a sealing or breathable tape to reduce the evaporation rate. A sealing tape can be used in biochemical assays, whereas a breathable sterile tape that does not obstruct gas exchange can be utilized in cell-based assays.
  • Utilization of low evaporation lids that have been specifically designed to minimize evaporation. Notably, low evaporation lids intended for use in cell-based assays have been designed to enable gas exchange. One of the companies that has pursued this approach is Wako Lab Automation, which has developed automated cellular and compound microplate lids, minimizing, and virtually eliminating the edge effect.