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The Right Reader

With an ever-increasing number of multimode microplate readers on the market, it is more important than ever to choose the right solution for your laboratory and workflow.

by Michael Fejtl
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Microplate reader requirements for cell-based assays need to be considered carefully

Having a reader that is poorly suited to your assays can be a real headache for researchers, causing the loss of valuable data and bottlenecks in your workflow, which can ultimately lower the productivity of the laboratory. In contrast, the careful selection of the right microplate reader can be a real boon for your cell-based applications, allowing faster, easier processing of samples and potentially enabling previously unachievable or impractical experiments to be performed.

What are you measuring?

The first decision to be made is the measurement modes you are likely to require. Fluorescence intensity measurement capabilities are vital for the majority of cell-based measurements, but an increasing number of assays now rely on luminescence-based or timeresolved techniques. While the ability to easily switch between measurement modes may not be important for a high-throughput facility using a reader for a single application, this flexibility is a key requirement for research centers. If your budget does not currently stretch to a system that gives you the level of flexibility you desire, a modular instrument may be the best solution, allowing you to purchase a system suitable for your current workload with the option to upgrade to additional measurement modes as necessary.

Similarly, instruments with filter-based optics are well suited to high-throughput applications requiring a single measurement type to be performed repeatedly, but monochromatorbased systems generally offer greater versatility by negating the need to purchase numerous filter sets to meet changing assay requirements. In either case, an instrument with the optics located below the microplate is vital for cell-based applications, as the volume and optical properties of the medium can significantly impact measurements from above the microplate, leading to poor reproducibility and a loss of data. This is particularly important for 3-D cell cultures, which have a greater depth of field. Instruments routinely used for cell-based assays ideally should have an adjustable Z focus, enabling the user to optimize the measurement height within the well for each assay to maximize sensitivity. Some systems also now offer fully automated Z focusing, further accelerating cell-based assays by determining the optimal Z position based on measurement of a reference well or multiple wells.

The size of the excitation apertures is also an important consideration for cell-based measurements, as the reporter molecule of interest is unlikely to be uniformly distributed across the entire well. Ideally, the light source should illuminate the entire culture at once, speeding up measurements and improving reproducibility by avoiding the need for multiple “spot” measurements at different points within each well. Autofluorescence from the growth medium is another significant issue for many cell-based assay systems, making some form of background signal correction vital to avoid small changes in cellular response being masked by noise from a high background signal. This autofluorescence, combined with the high cell densities encountered in some cell-based systems, can also lead to large changes in signal intensity across a measurement series, making it important to choose an instrument with a large dynamic range to avoid loss of sensitivity or data at one or both ends of the assay sequence.

What assay conditions do you need?

The optimal culture conditions will vary significantly with the cell line, assay type, and experimental duration, but rigorous environmental control is vital for any cell-based application. Almost all microplate readers now offer some form of temperature regulation, but as even small variations can lead to experimental bias or erroneous results, it is important to choose a reader with good thermostatic control. The measurement chamber of some readers can suffer large fluctuations over time as the system responds too slowly to increases or falls in ambient temperature. The location of the heating element can also affect assay performance, with uneven heating leading to temperature gradients across the plate, compromising both cell growth and enzymatic activity. As the experimental durations increase, this effect can seriously impact results and is potentially further compounded by differing evaporation rates between wells, leading to the formation of concentration gradients across the plate.

For many cell-based assays, effective control of gas pressure is also an important consideration. Maintaining in vivo-like partial pressures of CO2 and O2 can be vital for cellular survival and proliferation, particularly of eukaryotic cells, as many culture systems depend on precise regulation of atmospheric CO2 levels to control the pH of bicarbonate buffer systems. In addition, the hypoxic conditions found in vivo can significantly influence cellular regulation and responses. Most currently available microplate readers do not offer the ability to control CO2 and O2 partial pressures within the measurement chamber, and so culture plates must be regularly transferred between an incubator and the reader for measurement.

Although this is possible with some robotic systems, the added expense and complication are beyond the scope of most laboratory setups, leading to overnight gaps in data. In addition, the “shock” experienced by cells as they are regularly exposed to atmospheric conditions for measurement can bias results and hide subtle trends. Ideally, the reader should offer precise regulation of both CO2 and O2 within the measurement chamber, allowing continuous measurement without affecting results.

How does it fit into your workflow?

A reader that is poorly matched to your cell-based assay workflow can cause significant bottlenecks in processing and analysis, leading to a backlog of plates for measurement and potentially impacting time-critical experiments. For higher-throughput applications, choosing an automation-friendly instrument that can be integrated with your laboratory’s liquid-handling systems, downstream analytical devices, and LIMS can streamline analysis and data transfer while virtually eliminating the risk of transcription errors.

Cell-based assays offer significant advantages for many applications, providing greater biological insight than straightforward biochemical assays. Implementation of cell-based assays requires careful consideration of your reader requirements so that you select the system with the hardware and software that best fit your cell culture system and assay needs. Ideally, the readers should offer walkaway automation of assays, providing rigorous environmental control and allowing long-term, continuous measurement of cellular activity within the measurement chamber.