Which is the best microplate for my assay?

The selection of the optimal or wrong microplate, also called microtiter plate, can significantly affect the performance of your assay. In these few lines, I will give you general guidelines that should help you choosing the best microplate for your assay.

Dr Tobias Pusterla Dr Tobias Pusterla (10)

The microplate is probably the most underrated piece of equipment by scientists running microplate-based detection assays. It is usually considered as a passive support or container of the liquid that has to be measured.

Fig. 1: Microplates

Microplates: struggling through a multitude of choices

Although there are small differences in size and well position for different plate types and manufacturers, all microtiter plates come with the same footprint dimensions. These were defined by the SLAS (Society for Lab Automation and Screening), former SBS (Society for Biomolecular Screening).

Besides their density (number of wells/plate), microplates come in different colours, and with different volume capacities. With this plenty of options, it is sometimes hard to pick the optimal one for a specific assay.

Quite often, we as insiders tend to take this matter as obvious or self-explanatory. Recently, I have given a few talks providing general optimization guidelines for microplate reader-based detection. These included as well the choice of the right microplate. To be honest, at first I was quite embarrassed during my talks because I assumed I was exposing a no-brainer. Actually, I was wrong!

Hence, I thought that putting a brief summary on the multiwall plate matter on paper could possibly help a few people.

Which plate colour fits my assay at best?

Let´s start with the colour. Microplates come generally in four colours: clear, black, white, and grey. Transparent microplates are required for absorbance assays as light has to pass through the sample in this detection mode. Although this is quite easy, you have to consider that standard clear microplates made of polystyrene do not transmit wavelengths in the UV range (<320 nm). Hence, these microplates are well-suited for absorbance detection in the visible range like for colorimetric assays (such as ELISAs), but are totally inappropriate for RNA and DNA quantification. For this purpose, UV-transparent microplates that allow UV light transmission in the range between 200 – 400 nm have to be used. Depending on the manufacturer, these microplates have different names but they are all made of the same material, cyclic olefin copolymer (COC).

Black microplates partially quench the signal of the sample. This happens because the black colour partially absorbs the light signal coming from the liquid. Consequently, these plates are well suited for fluorescence intensity including FRET, and for fluorescence polarization assays. These are usually detection modes with a high signal yield, and the use of a black microplate helps reducing background, auto-fluorescence and well-to-well crosstalk, providing better signal/blank ratios.

Because of these very reasons, black microplates are generally not recommended for luminescent and TRF assays, as these usually have a low signal yield that would be further quenched by the black microplate. For these assays white plates a recommended. The white colour of the well reflects part of the sample signal, maximizing it. The drawback is that white microplates will also increase the blank signal. However, this is usually quite low in luminescent assays. In TRF, the delayed measurement window eliminates the influence of the background.

An in-between solution between black and white are grey microplates. These are specifically recommended for AlphaScreen® and AlphaLISA® as they are said to reduce cross-talk and background while still providing high signals.

Dedicated microplates for cell-based assays

When running bioassays or cell-based assays, detecting from the bottom of the well is usually recommended for several reasons. This obviously cannot be done in standard white or black plates. Microplates with a clear bottom have to be used. Depending on the assay the walls of the wells can be white or black. The clear bottom of these microplates can be of different materials from plastic (polystyrene or COC), up to glass or quartz. Although, the performance is significantly higher with glass, even more with quartz over plastic, the price for one single microplate is also significantly higher. Moreover, glass and quartz plates are not disposable and have to be cleaned and autoclaved in order to make them sterile after every use. Hence, one has to decide if the better performance that comes at a higher cost and with more maintenance, justifies the use of such a plate compared to a cheaper, easy-to-use microplate with clear plastic bottom.

Plate formats: is miniaturization good for me?

The well volume is the second point to be considered. The most common microplate format used is the 96-well one (up to 300 µL/well). Higher density microplate formats are usually employed for miniaturization purposes. Besides having more samples on the same microplate, using 384- (up to 100 µL/well), low volume 384- (up to 40 µL/well), 1536- (up to 15 µL/well), and even 3456-well formats (up to 5 µL/well) reduces the volume of reagents used and consequently the costs. A solution in-between, are half area 96-well microplates (up to 150 µL/well). Although they still bear 96 wells, their capacity is comparable to a 384-well plate (up to 100 µL/well).

With 384-well microplates the difference in volume affects also the shape of the wells. While regular 384-well plates have square-shaped wells, low-volume have round ones. Besides the volume, the difference in shape affects also other factors like shaking efficiency and meniscus. Of notice, since square wells share common walls with the neighbouring wells, they should be avoided in luminescence assays as they could generate severe well-to-well (through the wall) crosstalk.

Generally, the drawback of higher density microplates is that 1536- and 3456-well formats cannot be pipetted by hand, a pipetting machine is needed. For 384-well microplates manual pipetting is still doable but quite tedious and usually not recommended. Consequently, one has to weigh whether the higher handling costs associated to working with 1536- or 3456-well microplates are counterbalanced by the cost savings achieved by the miniaturization.

In the opposite direction, when greater volumes are required, 6-, 12-, 24- or 48-well plates can be used.

The very last thing: as a general rule, the lowest volume recommend for a microplate well in order to have an efficient and realistic measurement is generally >1/3 of the maximum volume of its well. Hence, for a standard 96-well microplate, you should not go below 100 µL.


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