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Tutorials : Jan 1, 2011 (Vol. 31, No. 1)

Increasing Throughput in Cellular Assays

Reduction of Edge Effect Allows Results to Remain Consistent Across Entire Plate
  • Tina Marwood
  • ,
  • Chandrasekaran Vasudevan
  • ,
  • Thomas Brevig

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.

Methods

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.

Results

Well-to-Well Variation in Cell Growth. As shown in Figure 1, the Edge plate with a filled moat has a cell count per field of 130–170 across the entire plate, whereas the Edge plate with an empty moat has a cell count per field of 0–130 cells in the outer wells, in comparison with 130–170 in the inner wells. Thus the Edge plate with a filled moat shows a much more even distribution of cell count per field across the entire plate.

Evaporation from Plates. The overall plate evaporation from six different plate types is shown in Figure 2. The presence of the evaporation buffer zone in the Nunc Edge Plate dramatically reduces the overall plate evaporation to less than 1% after four days incubation, or 2% after seven days incubation, in comparison to more than 8% for the other commercially available plates.

Effect of Agarose in the Moat. An evaporation buffer zone consisting of 0.5% agarose provides the same low plate evaporation as water, while providing the solidifying effect required to prevent spillages during automation. Both remain below 2% after seven days incubation, in comparison to over 8% observed with a standard 96-well plate (data not shown).

Field Focusing. When imaging multiple cellular targets in high-content analysis, it is vital that as many fields as possible are properly focused. Figure 3 demonstrates the percentage of poorly focused fields, where focusing was observed to be significantly better with the Edge plate than the other plates.

Conclusion

With the need to streamline processes by increasing throughput and automating protocols, it is vital that cell-culture plates are able to provide consistent and reliable results. Thus, the prevention of variation between the plates’ outer and inner wells allows results to remain more true to the population phenotype for more efficient cellular analysis.

The Thermo Scientific Nunc Edge Plate greatly reduces the edge effect, which is commonly experienced during cell culture. By reducing well-to-well variability, results remain consistent across the entire plate, enabling the confident use of the outer and corner wells to increase throughput. The reduction in evaporation observed enables concentrations to remain consistent across all wells and efficient cell growth across the entire plate. Furthermore, the ability to fill the moat with agarose provides a degree of solidity, preventing spillages upon loading to automated platforms, while maintaining the same effect as obtained with water.

Many cell-based assays require multiple targets to be simultaneously imaged, where all of the fluorescence from a single location needs to focus at the same point within the imaging system. Nunc Edge Plates are flat, minimizing the refocusing needed when sampling several fields per well, providing efficient automated imaging. As a result, users performing various high-content analyses can prepare cell cultures that remain healthy and viable across the Nunc Edge plate, while being imaged with ease.