Microplates For Luminescence Assays

Figure 5: Photo from the bottom of a 96-well ViewPlate microplate (left) and a 96-well clear-bottom IsoPlate microplate (right). The ViewPlate microplate has a one-piece rectangular clear plastic base on the underside of the plate. The IsoPlate microplate has individual circular clear plastic well bottoms that are planar with the bottom of the white- or black-framed wells.  

Tips and FAQS

• BackSeal plate seal can be used to convert a clear-bottom plate into an opaque (solid-colored) plate for reads. BackSeal plate seal is offered in both white and black (catalog number 6005199 for white, catalog number 6005189 for black). The color of the BackSeal plate seal chosen should match the color of the sides of the wells of the plate.

Q. What kind of plate seal can I use for my plates?

A. Seal-A adhesive plate seal (catalog number 6050185) can be used to prevent evaporation during incubation steps or during a luminescent plate read.

Q. What kind of lids can I use for my plates? 

A. Clear, sterile lids to fit our microplates can be ordered separately For 96-well plates, the catalog number for the lid is 6005619, and for 384-well and 1536-well plates the catalog number for the lid is 6007619. Lids for 96-well plates have condensation rings that align with the underlying wells. These lids will leave a small space between the lid and the well. This is necessary so cells can ‘breathe’ when growing. Lids should be removed prior to reading.

Microplates for coated-plate assays (including chemiluminescent ELISAs)

Sometimes referred to as "solid phase assays", coated-plate assays require the anchoring of one of the assay components (protein, antibody, sample, etc.) to the surface of the microplate. Coated-plate assays use wash steps to separate bound (associating) and unbound (non-associating) reagents from the well of the plate.

OptiPlate™ HB (high-binding) plates

High-bind OptiPlate microplates are uncoated plates that are specially treated to allow passive, direct coating of antibodies, proteins, samples, and other biomolecules using standard plate coating procedures. These plates are offered in black or white. They are solid (opaque) in color with no transparency, and therefore require measurement in -reading microplate readers.

IsoPlate™ HB (high-binding) plates

High-bind IsoPlate microplates are uncoated plates that are specially treated to allow passive, direct coating of antibodies, proteins, samples, and other biomolecules using standard plate coating procedures. The bottom of the wells is clear, while the sides of each well are either black or white in color. The clear-bottom base of the plate allows for bottom-read measurements (i.e., when the plate detector is located below the plate within the plate reader). It is always possible to convert an IsoPlate microplate into a completely solid-color plate to facilitate -reading measurements (i.e., detector is located above the plate within the instrument) by using BackSeal plate seal. BackSeal plate seal is offered in either black or white and is an adhesive sticker-like seal that is affixed to the underside of the plate. 

Selection table for coated-plate luminescent assays 

Tips and FAQS

• BackSeal plate seal can be used to convert a clear-bottom plate into an opaque (solid-colored) plate for reads. BackSeal plate seal is offered in both white and black (catalog number 6005199 for white, catalog number 6005189 for black). The color of the BackSeal plate seal should match the color of the sides of the wells of the plate.

Q. What kind of plate seal can I use for my plates?

A. Seal-A adhesive plate seal (catalog number 6050185) can be used to prevent evaporation during incubation steps or during a luminescent plate read.

Q. What kind of lids can I use for my plates?

A. Clear, sterile lids to fit our microplates can be ordered separately For 96-well plates, the catalog number for the lid is 6005619, and for 384-well and 1536-well plates the catalog number for the lid is 6007619. Lids for 96-well plates have condensation rings that align with the underlying wells. These lids will leave a small space between the lid and the well. This is necessary so cells can ‘breathe’ when growing. Lids should be removed prior to reading.

Q. Do you have any suggested plate coating protocols that I can use to bind my antibody/sample to the plate?

A. High-bind plates can be coated using any standard plate-coating method. Plates can be coated passively using the basic outline below:

1. Antibody, protein, or sample (concentration of ~10 µg/mL or higher) is incubated in the plate overnight in a carbonate buffer at an appropriate temperature (room temperature or 4 degrees Celsius). Select a temperature that will help maintain stability of the antibody, protein, or sample being coated.
2. Plate is washed three times with buffer (for example, 1X PBS).
3. Plate is "blocked" overnight to cover the well surface area that remains (typically using BSA, sugars such as trehalose or casein, serum, etc.)
4. Final washes are performed with buffer before using the plate in an assay.

For more information on plate coating, blocking, and storage, we recommend this reference:

Brown, M. C. (2011) Microtiter Plate Elisa, in Immunoassays in Agricultural Biotechnology (ed G. Shan), John Wiley & Sons, Inc., Hoboken, NJ, USA. doi: 10.1002/9780470909935.ch4 

Crosstalk Studies

ATPlite

Luminescence assays are usually measured in white microplates, since the white color reflects the light to give a maximal signal, whereas black microplates absorb some of the light and give a lower signal. In comparison to other assay technologies, luminescence assays are capable of generating relatively high signal levels, so well-to-well cross-talk may potentially be an issue.

In order to compare the signal levels and cross-talk generated using different colors of microplates, an ATPlite 1-step assay was performed using a high concentration of ATP. We compared white, black and light gray plates in 96- to 1536-well plate densities. In 96-well format we also looked at black & white IsoPlates, which are microplates that have white wells within a black frame.

Cell-based luminescence assays can be run in either clear-bottom or opaque plates. We measured the signal and cross-talk in clear bottom white and black ViewPlate microplates.

In this study the cross-talk reported is the average amount measured in the four wells adjacent to the well containing the ATPlite signal. The formula used to calculate the cross-talk and platemap used in the calculation are shown below. 

% Cross-talk = (Average cross-talk signal – Average blank)/(ATPlite signal – Average blank)*100
 

Figure 6. Platemap for calculating luminescence cross-talk Alpha Technology

Materials and Methods

Equal volumes of 1 micromolar ATP dissolved in PBS and ATPlite 1-step reagent were added to the microplate and mixed for 5 minutes using a plate shaker. The total assay volumes used for the different plate formats were:

1536-well – 5 µL 
384-well standard – 50 µL 
384-shallow well – 10 µL 
96-well – 200 µL

The volumes used are representative of typical assay volumes for these plate types. The plates were dark-adapted for 10 minutes and then read on the EnVision® multimode plate reader using ultrasensitive luminescence detection with a 0.1 second read time for opaque plates and 384-well ViewPlate microplates. 1536-well ViewPlate microplates were read using standard luminescence detection.  

Results

The % cross-talk calculated for each plate density is shown below. 

1536-Well

384-Shallow well

384-Standard well

96-well

Summary 

White plates give higher signal levels than black plates, but this is also accompanied by higher well-to-well cross-talk. The amount of cross-talk increased as the plate well-density increases. Cross-talk is highest in 1536-well plates, and less so in 384-well plates.

Cross-talk is not significant in either 384-shallow well or 96-well plates. These two types of plates are constructed so that the wells are connected by only a narrow piece of plastic.

If cross-talk is an issue using a white plate, switching to a gray plate may be preferable to using a black plate, since cross-talk is significantly reduced without a major loss in total signal. 

Alpha Technology

White microplates rather than black plates are usually recommended for luminescence assays, since the white color reflects the light signal, resulting in a higher total assay signal. However, at high signal levels or high plate well-densities, i.e. 1536-well plates, some of the light may be transmitted through the walls of a well and be detected in an adjacent well. Switching from a white plate to a black plate can reduce the amount of cross-talk, but at the same time the overall assay signal will be significantly reduced. As an alternative to using black plates to reduce well-to-well cross-talk, Revvity has developed light gray AlphaPlate™ microplates. The advantage of AlphaPlate microplates compared to black plates is that cross-talk can be reduced with much less reduction in to total assay signal.

We have conducted a study comparing the performance of AlphaScreen assays in white OptiPlate microplates to AlphaPlate microplates in various plate well-densities. AlphaScreen Omnibeads™ were used to generate the signal that was detected using the EnVision plate reader. The total signal and the well-to-well crosstalk were compared for the two plate types. In addition, we also compared the Z’-factor in white OptiPlate vs. AlphaPlate microplates. 

Alpha assays such as AlphaLISA™, AlphaLISA ™  SureFire® Ultra™, and AlphaScreen ™  produce a luminescent output signal when Donor beads that are excited at 680 nm transfer energy mediated by singlet oxygen to Acceptor beads in close proximity. When singlet oxygen comes in contact with Acceptor beads, an energy-transfer cascade is initiated that culminates in light output at 520-620 nm for AlphaScreen and 615 nm for AlphaLISA assays. When using Revvity’s EnVision™  or EnSpire™  multilabel readers the excitation energy source is a high power laser. The power level of this laser is considerably higher than that of a standard flash lamp used as an excitation source for other detection technologies. 

Materials and Methods

A solution of Omnibeads was prepared by adding 100 µL of Omnibeads to 25 mL of PBS. The Omnibead solution was added to the high signal well and PBS to the other wells of a white OptiPlate microplate and the corresponding AlphaPlate microplate. The volumes used for the different plate formats were:

• 1536-well – 5 µL
• 384-well standard – 20 µL
• 384-shallow well – 10 µL
• 96-well ½ AreaPlate microplate – 50 µL

The volumes used are representative of typical assay volumes for these plate types. The plates were read on the EnVision® multimode plate reader using the standard factory-defined AlphaScreen settings.

An example of the AlphaScreen cross-talk pattern that is typically seen is shown in Figure 7. The well in the center of the grid was filled with a solution of Omnibeads, and all other wells with PBS. This specific data was generated in a 1536-well OptiPlate microplate; however, the same general pattern is seen in other plate well-densities. The two adjacent wells in the same row as the well containing OmniBeads show the greatest amount of cross-talk; lower cross-talk is also seen in adjacent wells in the rows above and below.