A Simple and Cost Efficient Method to Avoid Unequal Evaporation in Cellular Screening Assays, Which Restores Cellular Metabolic Activity
Angelika Walzl, Nina Kramer, Giulia Mazza, Margit Rosner, Dieter Falkenhagen, Markus Hengstschläger, Dagmar Schwanzer-Pfeiffer, Helmut Dolznig

Abstract
Spheroid based cellular screening approaches represent a highly physiologic experimental setup to identify potential novel chemical compounds in cancer research. Increasing numbers of chemical compound libraries are available, which offer straightforward development of preclinical drug screening assays for academic research. Cellular screening experiments are often performed in 96 well plates in a small culture volume for elongated time periods; therefore, growth medium volume decrease due to evaporation poses a problem. We show here that uneven loss of growth medium depending on the microwell position on the plate (edge and corner versus central positions) led to significantly different readouts in a cell metabolism assay (alamarBlue®) as indicator for cellular health and proliferation. These effects are due to both an increase in dye concentration and reduced cellular metabolic activity owing to the decrease in culture volume. The incubation of the 96 well plates in cheap, custom-made humidity chambers almost abolished the loss of liquid and restored equal growth medium volumes in all wells after prolonged culture. This led to evenly distributed alamarBlue® fluorescence intensity values on the multiwall-plate and warrants proper identification of effective compounds in screening assays.

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