January 1, 2011 (Vol. 31, No. 1)
Reduction of Edge Effect Allows Results to Remain Consistent Across Entire Plate
With significant resources and money being invested in the identification of lead compounds in the drug screening process, it is critical that researchers have a highly efficient, reliable, and often automated method of culturing cells for various cellular assays. As such, the culture plate and its ability to integrate with automated systems is key to streamlining an effective high-throughput process.
However, the commonly experienced “edge effect” can often impact on data consistency. The evaporation of medium from wells during incubation is especially prominent in the wells closest to the perimeter of the plate (the edge wells) and results in well-to-well variations. As medium evaporates, concentrations are consequently altered, and cell growth is adversely affected.
Differential evaporation across the plate results in variability; a volume loss as small as 10% concentrates media components and metabolites enough to alter cell physiology and, in some cases, this can be quite severe. Furthermore, plates are often not optimized for imaging purposes, making it difficult to obtain clearly focused fields. As a result, the plate cannot be used efficiently.
In an attempt to alleviate the edge effect, researchers often decide not to culture cells in the outermost wells but to fill these with sterile water, and use only the inner wells of each plate for cell cultures. By rendering these wells unusable, throughput and, therefore, efficiency, is substantially reduced. The additional difficulties of obtaining clearly focused field images also hinders throughput since multiple screenings may need to be performed in order to obtain usable results.
Thermo Fisher Scientific recently developed a novel plate design that incorporates a large evaporation buffer zone (or moat) built into its perimeter, which can be filled with sterile water. Alternatively, through the inclusion of 0.5% agarose, for example, the moat is provided with a solid, jelly-like material to eliminate spillages, making it usable as a stand-alone plate or as part of a fully automated workflow. In this article, the effects of the moat on well-to-well variability in cell growth and overall plate evaporation are reviewed. In addition, the effect of an extremely flat plate on eliminating blur in imaging applications is investigated.
Four different methods were performed to determine well-to-well variability in cell growth, overall plate evaporation, the effectiveness of agarose in enabling workflow automation, and efficiency of field focusing. The protocols were carried out using a variety of commercially available 96-well plates.
Cell Growth. Two plate types—a Nunc™ Edge plate with sterile water in the moat and a Nunc Edge plate with an empty moat—were used to culture HeLa cells. The cells were cultured in an incubator at 37ºC and 5% CO2. After seven days of culture, the cells were fixed with formaldehyde and stained with fluorescent dyes for visualization. The plates were then imaged with a high-content screening reader using a 10x objective and cell count data was collected.
Evaporation. Six plate types were filled with 100 µL of water. The plates were subsequently incubated at 37ºC for four days in a humidified atmosphere of 5% CO2 in air. In order to simulate common laboratory conditions, the incubator door was opened for 15 seconds, seven times each day. To compensate for favorable versus nonfavorable positioning within the incubator, three plates of each type were placed at different positions and a mean value calculated. The protocol was repeated over an incubation period of seven days using 200 µL of water.
Moat Filled with Agarose. Three plate types—an Edge plate with water in the moat, an Edge plate with 0.5% agarose in the moat, and a standard 96-well plate—were filled with 100 µL of water and incubated as described previously.
Field Focusing. Eleven wells per plate, from three different plate types, were imaged with a 10x objective to obtain 45 fields per well. Only the first field image in each well was obtained using the autofocus function. Upon visual inspection, each field was determined to be either in sharp or poor focus. Results from all three plate types were subsequently compared to calculate the percentage of fields that were in poor focus.
Tina Marwood (email@example.com) is research manager, Chandrasekaran Vasudevan is platform manager, detection, and analysis, and Thomas Brevig is director, global research, labware, and specialty plastics.