Taming Standard Deviation in Cell-based Assays

Some researchers experience “edge-effects” in their assay plates, while others don’t seem to have this problem. Edge-effects are characterized as a significant relative reduction in measured RLU signals from the edge wells of the assay plate. The phenomenon is a bit of a “black box”, but it almost certainly results from the local cell culture environment. One common belief is that differential rates of evaporation across the assay plate occur during the assay incubation period.  Because of the air movement within mammalian cells incubators, there is flow of warm air moving across the assay plate, and edge wells suffer from a higher rate of evaporation, and evaporative cooling, due to their immediate proximity to the outside environment.  We believe this is a plausible contributing factor to the occurrence of edge effects, and there are some techniques worth trying to potentially combat it.

One common method that may help to mitigate edge effects is to dispense sterile water into the inter-well spaces of the assay plate. Doing so nearly doubles the liquid mass of the plate, greatly increasing the temperature buffer and vapor source under the plate lid. Also, we recommend that researchers go to extreme measures to ensure that their 37°C/CO₂ incubators maintain a very high internal humidity, to the point of seeing condensation on the inner glass door. All of INDIGO Biosciences’ incubators have at least two water pans, which provide ~70-80% relative humidity around the assay plates. Be aware that every time the incubator door is opened warm, humid air escapes the incubator, resulting in cycles of internal cooling followed by forced heating and re-humidification. In an ideal lab environment, researchers will dedicate an incubator to their assay plates. Once your plates are in, close the door and don’t open it until the assay endpoint. This, of course, can be difficult if one’s colleagues are going in-and-out of that same incubator to add or retrieve their assay plates during your incubation period. Keep in mind that heavy incubator traffic will be to the detriment of your assay results.

Alternatively, a commonly used remedy to edge effects is to simply avoid using the outside wells of an assay plate. Sacrificing edge wells is never a desirable choice, but if your edge-effect problem persists, we suggest that you consider adopting that strategy. If you avoid using the outside plate wells in your assay setup, be sure to dispense sterile water into them so that they will continue to contribute a vapor source to the plate and a thermal buffer to the internal wells.