Microplates For Absorbance and Colorimetric Assays

Example of a colorimetric ELISA assay.

Plate considerations

Absorbance wavelength and plate spectral properties

Assays that measure absorbance in the visible light range (400-900 nm wavelengths) can be run in clear-bottom polystyrene plates. Assays that measure absorbance in the ultraviolet range (200-400 nm wavelengths) will require plates that are made of a UV-transparent material (like glass or Cyclic Olefin Copolymers), to avoid absorbance of light by the plastic itself.

Plate considerations

Assays that measure absorbance in the visible light range (400-900 nm wavelengths) can be run in clear-bottom polystyrene plates. Assays that measure absorbance in the ultraviolet range (200-400 nm wavelengths) will require plates that are made of a UV-transparent material (like glass or Cyclic Olefin Copolymers), to avoid absorbance of light by the plastic itself.

Signal

Most absorbance (colorimetric) assays are run in clear plates, such as SpectraPlate™ microplates. This is because the clear color of the plastic should not interfere with measurements in the visible light range (400-900 nm wavelengths). Clear polystyrene plastic should not absorb light in the visible range.

Cross-talk

Cross-talk occurs when absorption from a neighboring well interferes with the measurement of the well-of-interest, and can sometimes be problematic in higher plate density formats (384-well, 1536-well). The use of clear-bottom plates that have white well walls (such as IsoPlate™ and ViewPlate™ microplates) can be helpful when cross-talk is an issue.

Microplates for cell-based absorbance assays

Choosing the correct microplate for a cell-based assay will be dependent on both the specific cell line being used in the assay and the assay protocol itself. Questions that need to be answered in choosing the plate include:

1. Do I need a sterile, tissue culture-treated plate?
2. Does the plate need to be coated?
3. Should the plate have a clear or opaque bottom?

Plate considerations

Whether or not it is necessary to use a sterile, tissue culture-treated plate depends on the length of time the cells are going to be in the assay plate. In some assays the cells are added to the microplate and the assay is completed within a few minutes to a few hours. In other cases, cells are grown in plates at least overnight prior to performing the assay or are treated with compounds for extended lengths of time. As a general recommendation, if the assay is going to be performed within a single working day a sterile, tissue culture-treated plate is not necessary. If the cells are going to be in the plate overnight or longer a sterile, tissue culture-treated plate should be used, and aseptic techniques should be followed.

The need for tissue culture-treated or coated plates depends on the specific cell lines used and how the cells are going to be treated in the course of the assay. Cells can be broadly divided into three classes of cells:

1. Strongly adherent cells
2. Poorly adherent cells
3. Suspension or non-adherent cells

Our sterile microplates are all tissue culture-treated to promote cell attachment and growth. The tissue culture treatment process involves exposing a polystyrene microplate to a plasma gas in order to modify the hydrophobic plastic surface to make it more hydrophilic. The resulting surface carries a net negative charge due to the presence of oxygen-containing functional groups such as hydroxyl and carboxyl. Strongly adherent cells will usually attach satisfactorily to tissue culture-treated plates. Cell lines that attach less strongly may require a plate with a coating such as poly-D-lysine or collagen which promotes attachment better than just tissue culture treatment. Cell-based assays using suspension cells are generally performed in standard tissue culture-treated plates. Coated plates are not typically used with suspension cells.

In addition to the specific cell line being used in the assay, the assay protocol itself is important in deciding the type of plate to use. For example, assays using adherent cells may include culture medium changes or wash steps in the protocol. In such cases it may be advisable to use a coated plate for the assay in order to prevent the cells from becoming detached from it during the assay.

Clear bottom vs. opaque plates

Microplates with clear bottoms can be useful for cell-based assays as they allow the microscopic visualization of the cells to monitor confluency, morphology and other parameters that may affect the cellular response in the assay. In addition, assays that are configured for bottom reading require clear bottom plates.

Clear bottom plates can be converted to functionally opaque plates by application of a BackSeal. BackSeals are available in either white or black (catalog number 6005199 for white, catalog number 6005189 for black). The color of the BackSeal should match the color of the sides of the plate wells.

SpectraPlate™ TC clear microplates

Not all SpectraPlate microplates are sterile/tissue culture-treated. Look for SpectraPlate products that are designated as "TC" (tissue culture-treated) for cell-based assays using adherent or suspension cells. SpectraPlate microplates are completely transparent and have no color. The clear-bottom base allows for microscopic visualization, which can be helpful when microscopic observation is required to check cell density and morphology. The clear-bottom base of the plate also allows for - or bottom-read measurements. These plates are offered in both 96-well and 384-well format.

Selection table for cell-based absorbance and colorimetric assays

Tips and FAQS

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

A. TC-treated SpectraPlates, as well as all TC-treated microplates from Revvity, are supplied with a lid. The exception to this is when the TC-culture treated plate is purchased in a case of 200. In this instance, lids are not supplied with the microplates and much be purchased 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 the plate to prevent damage to the plate reader.

Q. Can I use a seal on my plates, or will that kill my cells?

A. If cell viability is no longer an issue, Seal-A^™ adhesive plate seal, catalog number 6050185, can be used to prevent evaporation during incubation steps. The plastic of the Seal-A adhesive seal has some spectral properties that may interfere with assays performed at absorption wavelengths. It may be necessary to remove the seal from the plate prior to reading. If cell viability is an issue at the time a seal is needed, we recommend using sterile plate lids (if sterile practices need to be maintained) or breathable plate seal (if antibiotics/antifungals can be added to the culture media to prevent contamination). Breathable plate seals are available from various suppliers, including Nunc® and Corning®.

Microplates for biochemical absorbance assays

Microplates for standard, in vitro absorbance/colorimetric assays that do not require anchoring of cells or other reagents to the surface of the plate.

SpectraPlate microplates

SpectraPlate plates are completely transparent. The clear-bottom base allows for -read or bottom-read measurements. These plates are offered in 96-well, 384-well (standard or shallow well), and 1536-well formats.

ViewPlate^™ and IsoPlate^™ microplates

In cases where cross-talk may be an issue (particularly in higher density 384-well or 1536-well formats), ViewPlate or IsoPlate microplates with white-walled sides can be tested. These plates are offered with a clear-bottom base, with the sides of each well a solid white (or black) in color.

Selection table biochemical absorbance and colorimetric assay

Clear-bottom IsoPlate microplates are similar to ViewPlate microplates in that the bottom of the plate is clear, while the sides of each well are either black or white. This makes the IsoPlate microplate suitable for bottom-reading instruments. However, there are a few differences between IsoPlate and ViewPlate microplates. IsoPlates are manufactured by first molding 96 clear wells at a time, then molding a black or white frame around the clear wells. This makes the white- or black-colored well extend to the same depth as the clear well base and can help reduce cross-talk in bottom-reading assays. IsoPlates were originally developed for coincidence counting in a MicroBeta™  radiometric detection instrument (reading from top and bottom coincidentally). While they have clear bottoms, IsoPlates are not ideal for confocal imaging (microscopic observations) because the optical clarity of the bottom is not as good as clarity is with CellCarrier™ Ultra or ViewPlate microplates.

Tips and FAQS

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. The plastic of the Seal-A adhesive seal has some spectral properties that may interfere with assays performed at certain absorption wavelengths. It may be necessary to remove the seal from the plate prior to reading.

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

A. Clear, non-sterile lids that fit our SpectraPlate and ViewPlate microplates can be ordered separately. These lids will leave a small space between the lid and the well, which can lead to evaporation over longer periods of time. Lids should be removed prior to reading the plate to prevent damage to the plate reader.

Microplates for coated-plate assays (including colorimetric 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.

SpectraPlate HB microplates

High-bind SpectraPlate 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 completely transparent. The clear-bottom base allows for -read or bottom-read measurements. These plates are offered in 96-well and 384-well format.

Selection table for coated-plate colorimetric assays

Tips and FAQS

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. The plastic of the Seal-A adhesive seal has some spectral properties that may interfere with assays performed at certain absorbance wavelengths. It may be necessary to remove the seal from the plate prior to reading.

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

A. Clear, non-sterile lids that fit our SpectraPlate microplates can be ordered separately. These lids will leave a small space between the lid and the well, which can lead to evaporation over time. Lids should be removed prior to reading the plate to avoid damage to the plate reader.

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 °C). 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

Plate seals

Revvity offers a variety of plate seals. Seal™ is a range of plate seals that are applied to the surface of the plate and are used to prevent evaporation or radioactive contamination during assay incubation steps and/or plate reading measurements. Seal-A can be left on the plate during luminescence, AlphaScreen™, AlphaLISA™, AlphaLISA® SureFire® Ultra™, and radiometric measurements. Seal™ plate seals have spectral properties that may interfere with other types of assay measurements (absorbance assays, colorimetric assays, fluorescence assays). For these types of assays, you should compare the plate measurement with and without a Seal plate seal to test for interference. BackSeal plate seals are plate seals that are applied to the bottom of the plate. BackSeal plate seals can be used to seal the bottom of a filter plate prior to the addition of scintillation cocktail, preventing leakage. BackSeal plate seals can also be used to change a clear-bottom plate into a white- or black-bottom plate to reduce cross-talk during -reading measurements.

Plate Seal Products

Custom plate services at Revvity

• Revvity offers custom microplates services, including:
• Bulk ordering
• Fast and flexible plate barcoding
• Biological plate coating including – poly-D-lysin, collagen, streptavidin, and antibody coating
• Custom treatments including – tissue-culture, high protein binding and low protein binding
• Customer sterilization

For research use only. Not for use in diagnostic procedures.

The information provided above is solely for informational and research purposes only. The information does not constitute medical advice and must not be used or interpreted as such. Consult a qualified veterinarian or researcher for specific guidance or use information. Revvity assumes no liability or responsibility for any injuries, losses, or damages resulting from the use or misuse of the provided information, and Revvity assumes no liability for any outcomes resulting from the use or misuse of any recommendations. The information is provided on an "as is" basis without warranties of any kind. Users are responsible for determining the suitability of any recommendations for the user’s particular research. Any recommendations provided by Revvity should not be considered a substitute for a user’s own professional judgment.