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Microplates For Absorbance and Colorimetric Assays

Overview


Absorbance and colorimetric assays are designed to detect or quantitate the amount of a particular reagent in an assay by measuring the amount of light absorbed by the reagent or chromogenic reaction product at a characteristic wavelength. This wavelength is specific to the reagent being measured. The greater the amount of reagent there is present in the well, the more light that will be absorbed. Many colorimetric assays utilize a "chromogenic" (color-producing) substrate that, when converted into its final product, will absorb light at a specific wavelength.

Examples of absorbance assays include colorimetric ELISAs (such as the Alliance p24 kit), ELAST ELISA, assays that use chromogenic substrates (such as BCIP, DAB, 4CN, and Fast Red), Bradford assays, assays designed to quantitate based on the molar extinction coefficient of a substance, and other absorbance and colorimetric assays.

Colorimetric ELISA assay
Example of a colorimetric ELISA assay.

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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 COC), 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.

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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 the plate during the assay.

See additional information on plate treatments and coatings.


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 top- or bottom-read measurements. These plates are offered in both 96-well and 384-well format.

Selection table for cell-based colorimetric assays

Plate type

Plate properties

Notes

SpectraPlate TC clear microplates

Clear plate; sterile; tissue culture-treated for adherent cell attachment.

Can be used for either top-reading or bottom-reading microplate readers; helpful when microscopic visualization is required to check cells. Offered in 96-well or 384-well format.



Tips and FAQS

Q. What kind of lid can I use for my plates?
A. SpectraPlate TC plates are available with or without lids. Alternatively, clear sterile lids can be ordered separately (catalog number 6005619 for 96-well plates, catalog number 6007619 for 384-well and 1536-well plates). 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, TopSeal-A™ adhesive plate seal, catalog number 6050185, can be used to prevent evaporation during incubation steps. The plastic of the TopSeal-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®.

Q. How do I pipette my cells into microplates under sterile conditions? Can I continue to culture them in microplates?
A. We have a video tutorial on working with cells in microplates.

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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 MB microplates
SpectraPlate MB plates are completely transparent. The clear-bottom base allows for top-read or bottom-read measurements. These plates are offered in 96-well or 384-well format.


ViewPlate and IsoPlate microplates (untreated)
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 for biochemical colorimetric assays

Plate type

Plate properties

Notes

SpectraPlate MB microplates

Clear plate developed for colorimetric/absorbance assays.

Can be used for either top-reading or bottom-reading microplate readers. Offered in 96-well and 384-well format.

ViewPlate microplates

Clear-bottom plate (the bottom of the plate is clear, while the sides of each well are white)

Can be tested when using higher density plate formats (384-well, 1536-well formats) to reduce cross-talk. Offered in 96-well, 384-well and 1536-well format.

Clear-bottom IsoPlate microplates

Clear-bottom plate (the bottom of the plate is clear, while the sides of each well are white)

Can be used to reduce cross-talk; Only available in 96-well format

*Clear-bottom IsoPlate microplatess 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. IsoPlate plates 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. IsoPlate microplates were developed for coincidence counting in a MicroBeta® instrument (reading from top and bottom coincidentally). However, IsoPlate microplates are not ideal for confocal imaging (microscopic observations) because the optical clarity of the bottom is not as good as clarity is with ViewPlate microplates. Additionally, IsoPlate microplates are only available in 96-well format.


Tips and FAQS

Q. What kind of plate seal can I use for my plates?
A. TopSeal-A adhesive plate seal, catalog number 6050185, can be used to prevent evaporation during incubation steps. The plastic of the TopSeal-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 (catalog number 6005617 for 96-well plates, catalog number 6007617 for 384-well or 1536-well plates). 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.

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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 top-read or bottom-read measurements. These plates are offered in 96-well and 384-well format.

Selection table for coated-plate colorimetric assays

Plate type

Plate properties

Notes

SpectraPlate HB microplates

High-binding plate for direct plate coating protocols; clear plate developed for colorimetric/absorbance assays

Can be used for either top-reading or bottom-reading microplate readers. These plates are offered in 96-well and 384-well format.


Tips and FAQS

Q. What kind of plate seal can I use for my plates?
A. TopSeal-A adhesive plate seal, catalog number 6050185, can be used to prevent evaporation during incubation steps. The plastic of the TopSeal-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 (catalog number 6005617 for 96-well plates, catalog number 6007617 for 384-well or 1536-well plates). 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 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

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Plate seals


PerkinElmer offers a variety of plate seals. TopSeals are plate seals that are applied to the top surface of the plate, and are generally used to prevent evaporation or contamination during assay incubation steps. TopSeal plate seals have spectral properties that may interfere with certain types of assay measurements (absorbance assays, colorimetric assays, fluorescence assays). In these cases, you should compare the plate measurement with and without TopSeal plate seal to see if the seal can be left on the plate during measurement. BackSeal plate seals are applied to the bottom of the plate. These seals can be used to change a clear-bottom plate into a white- or black-bottom plate when needed.

 Table. Plate seal products

ProductType of sealPlate formatNumber of sealsCatalog number
TopSeal-A plate seal Clear adhesive seal  Any1006050185
BackSeal plate sealWhite adhesive seal(all)556005199
Black adhesive seal(all)556005189

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Guides


Microplate Catalog

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Custom plate services at PerkinElmer


PerkinElmer offers custom microplate services, including bulk ordering, fast and flexible plate barcoding, biological plate coating (including poly-D-lysine, collagen, streptavidin coating, antibody coating, and other coatings on request), custom tissue culture-treatment, custom high protein binding treatment, custom sterilization of microplates, special packaging, and other microplate treatments. If you are interested in custom plate services, please contact our custom service team:

ON>POINT® Custom Services

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