Problem: There is little doubt that cell-based assays are becoming a mainstay across the pre-clinical drug discovery process. Many of these assays are end-point based, where live cells are cultured in microplates, incubated with compounds of interest and then lysed to release the contents of the cell such that they can be assayed with reagents subsequently added to the wells. Often, the incubation times required for the assay can be counted in the minutes to decades of minutes, such that maintaining a controlled environment for cell health is not a significant concern. For those end-point assays that require longer incubation times of hours to decades of hours, controlled environments become a substantial concern and requirement. Depending on sample throughput required, this can be accomplished by manual intervention and placing the microplate(s) in a standard CO2 incubator or by robot into a microplate hotel.
Yet there is a growing trend in the use of live cell assays, where the cellular response to compounds is monitored kinetically through the use of non-toxic, cell membrane permeable reagents or phenotypic methods. This real-time response gives researchers an additional level of information that may be missed in single time point, end-point assay. While these live cell assays are easily incorporated into workflows for assays conducted over an hour or so, longer term responses can be problematic to obtain due to the difficulty in maintaining a controlled environment over the microplate to maintain cell health while kinetically reading the cellular response.
Solution: The addition of an atmosphere control unit to a microplate reader offers a solution to this problem by providing a controlled environment within the microplate reading chamber in which live cells can be nurtured. BioTek’s Synergy H1, for example, has a number of features that are important for successful live cell assays, including precise temperature control up to 45°C, controlled shaking of the microplate, and a gas controller module that allows control and monitoring of CO2 and O2 within the reading chamber. The precise control of both CO2 and O2 allows adjustment to the optimal physiological conditions up to 20 percent and down to nearly 0 percent for hypoxic and nearly anoxic conditions that many live cell assays require. Microplate readers with these features combined are ideal for kinetic live cell assays.
In particular, the Synergy H1 microplate reader has quadruple grating monochromator optics for fluorescence intensity measurements from the top or bottom of the microplate. UV-Vis and luminescence measurements can also be made with the system. The reader’s optics are modular, with an option to add filter-based fluorescence along with monochromator-based fluorescence.
To confirm the efficacy of the Synergy H1 system for live cell assays, mouse NIH3T3-GFP cells were compared using three microplate-based growth conditions:
• incubation in a dedicated tissue culture chamber with a manual transfer of cells to the microplate for analysis,
• incubation in the microplate reader chamber without CO2 control, and
• incubation in the microplate reader with 5 percent CO2 control and monitoring
Cell growth was monitored by GFP fluorescence detection, with readings taken every two hours for the manual method and every 30 minutes for the reader incubated methods over a total 70-hour runtime. As seen in Figure 1, data is expressed as a fold increase over time and shows comparable growth rates for manual and Synergy H1 equipped with the gas controller. The microplate reader method where CO2 was not used shows significantly lower cell growth.
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Comparison of mouse NIN3T3-GFP cell growth conditions.
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