Problem: Performing long-term, cell-based applications with living cells using a multimode microplate reader is becoming more and more popular in life science research. There are various absorbance-, fluorescence- and luminescence-based assays that require alternating cell incubation and signal detection over a long period of time–from several hours up to several days–including reporter assays, proliferation and viability studies, cytotoxicity and cell death studies, and many more. Besides optimizing the optical properties of the detection device, the major requirement for these applications is to keep cells viable and proliferating throughout the whole experimental period.

At present, a tedious transfer of the microplate between a common cell incubator and the microplate reader is the standard procedure, but has critical drawbacks. Without costly robotic solutions, the user has no choice but to manually transfer the plate for each detection step, increasing the risk of contamination and resulting in the loss of data, especially for overnight experiments. Furthermore, when incubating at low oxygen levels, transferring a plate from the incubator to the reader results in a change of environmental conditions, which can critically affect the outcome of the study.

Solution: The simplest solution to this problem is a microplate reader offering rigorous environmental control inside the measurement chamber, particularly the regulation of atmospheric gas concentrations.

For example, the Infinite® 200 PRO multimode reader is the first fusion of a microplate reader and a cell incubator on the market. Tecan’s patent pending Gas Control Module (GCM™) allows independent and simultaneous control of oxygen (0.1- 21 %) and carbon dioxide (0-10 %) concentrations inside the reader’s measurement chamber, with an altitude correction function to allow automatic compensation for variations in the atmospheric partial pressure of CO2. This ensures greater biological relevance for a wide range of studies, extending the experimental window for microplate-based investigations in proliferation studies (especially with anaerobe and facultative anaerobe bacteria), and allowing close replication of hypoxic and physiologic conditions for eukaryotic cells.

The GCM can also be used with pre-mixed gases, enabling flexible environmental control for a broad range of cell biology applications and ensuring excellent viability and proliferation for various cell types, throughout the whole period of analysis. The system’s process security features include both audible and visual warnings to alert the user of changes in gas pressure or flow, as well as continuous monitoring to easily identify if the target concentration will not be reached or deviates significantly during incubation.

Tecan has conducted several studies to evaluate the Infinite 200 PRO’s GCM-related functions, including proliferation of GFP-transfected human epithelial carcinoma cells inside the microplate reader over a period of 75 hours. In this study, cells incubated at five percent CO2 stayed viable and kept proliferating throughout the experiment, whereas cells incubated without CO2 regulation ceased proliferation and began to die within 24 hours. In a second experiment, it was demonstrated that hypoxia could be induced as efficiently in the Infinite 200 PRO as in a common CO2/O2 cell incubator, eliminating the need for a separate O2 incubator and offering complete workflow automation. Finally, it was demonstrated that growth studies with facultative anaerobe bacteria, such as Helicobacter pylori, can be quickly and conveniently performed. H. pylori cells were grown inside the reader at five percent O2, ten percent CO2 and 37°C for 27 hours, with OD60 measurements every hour.

To minimize evaporation during long term incubation periods inside the reader, Tecan recommends using Thermo Fisher Nunc™ Edge plates. The evaporation moats built into these plates provide passive humidity control, reducing evaporation to insignificant levels for incubation periods of up to three days. Cell biology-oriented functions, like those included in the Infinite 200 PRO, such as enhanced fluorescence bottom reading with Optimal Cell Reading (OR), for a better sensitivity and reproducibility in applications with adherent cells; efficient shaking capabilities, for a better optimization of the growth of suspensions cells; automated Z-focusing for top reading fluorescence measurements, to maximize signalto- blank ratios when working with colored cell media; and precise temperature control can help users set new standards for cell based assays.

For more information, visit www.tecan.com/gcm