Microplates For Fluorescence Assays
- Overview
- Black vs. white plates
- Microplates for cell-based fluorescent assays
- Microplates for biochemical fluorescent assays
- Microplates for coated-plate assays (including DELFIA TRF immunoassays)
- Application notes, posters and guides
- Plate recommendations for 爱游戏平台注册登录 fluorescent assays
- Plate seals
- Custom plate services at 爱游戏平台注册登录
Overview
Fluorescence is the emission of light by a substance, resulting from energy acquired by first exciting the substance using light at a different wavelength. In fluorescence assays, the chemical or dye that fluoresces is referred to as a "fluorophore". Each fluorophore has its own unique spectral properties; it will require excitation at a particular wavelength, and will release light at a particular emission wavelength. The excitation and emission wavelengths are not discrete wavelengths, but rather a range of wavelengths characteristic to a given fluorophore. In a typical fluorescent assay the fluorophore is excited by light at a given wavelength, and the signal at an emission wavelength is measured using a plate reader.
Figure 1. Excitation and emission spectra for a fluorescent DELFIA® Europium chelate.
Examples of fluorescence assays include fluorescence intensity (FI) assays, fluorescence polarization (FP) assays, FRET assays (Förster resonance energy transfer assays), fluorescent calcium flux assays, TRF assays (time-resolved fluorescence assays, including DELFIA), and TR-FRET assays (time-resolved Förster resonance energy transfer assays, including LANCE®).
Black vs. white plates
Autofluorescence
Autofluorescence is fluorescence resulting from substances other than the fluorophore-of-interest, and can negatively affect an assay by increasing background signal. Many components of assays buffers and biological samples can autofluoresce. Autofluorescence is triggered by the same excitation light used to excite the fluorophore in the fluorescence assay. The severity of background autofluorescence can vary based on the excitation wavelength being used in a particular assay. For example, higher excitation wavelengths (above 650 nm) usually cause less autofluorescence than wavelengths in the UV/Vis range.
Because white plates reflect light and black plates tend to quench light, background fluorescence will be higher in white plates as opposed to black plates. For this reason, black plates are typically recommended for fluorescence assays that use short half-life fluorophores. Time-resolved fluorescence assays, which use longer half-life fluorophores, can use either white or black plates (read below for more information).
General fluorescence assays (fluorescence intensity, fluorescence polarization, FRET)
General fluorescence assays include traditional fluorophores such as fluorescein, cyanine 3, cyanine 5, green fluorescent protein (GFP), rhodamine, Texas Red, coumarin, and other fluorophores. These fluorophores have relatively-short half-lives (< µsec, for microplate-based assays). We recommend running assays utilizing short half-life fluorophores in black plates to reduce background autofluorescence.
Time-resolved fluorescence assays (TRF, TR-FRET)
Time-resolved fluorescence assays involve fluorophores that have longer half-lives (µsec – msec, for microplate-based assays). Examples of such fluorophores include Europium chelates and cryptates, Samarium chelates, and Terbium chelates and cryptates. Because of the longer half-life of the fluorescent signal, you can set up your instrument to incorporate a "lag time" or "delay time" between the time the fluorophore is excited, and the time you begin reading the emission signal (time-resolved mode). This allows background autofluorescence to fade before you begin collecting emission signal from assays involving a long half-life fluorophore. Because of this, time-resolved fluorescence assays can be run in either black or white plates. The use of white plates will result in higher raw signals, because the light is reflected maximally by the white color of the plate. The use of black plates will result in lower raw signals, because the black color of the plate can quench the light. However, black plates may help in situations where cross-talk is an issue, resulting in better sensitivity. In general, we recommend white plates for higher-density time-resolved fluorescence assays (for example, assays run in 384-well and 1536-well plates) and other assays where a lower signal might be expected.
Figure 2. Measurement of a long half-life fluorophore. The fluorescent signal for this particular fluorescent reagent lasts longer than 1000 μs, allowing for a time-resolution read. The fluorophore is excited, which also produces background autofluorescence. This autofluorescence fades quickly in relation to the signal from the long half-life fluorophore, allowing you to use a delay time before collecting emission signal to avoid collecting signal from autofluorescence.
Plate density considerations
Fluorescent signal is higher in white plates because the light is reflected maximally by the white color of the plate. The use of black plates can quench the light, leading to a reduced signal. For assays using long half-life fluorophores (for example, DELFIA and LANCE assays), you could potentially use either white or black plates. However, as the plate density increases (384-well and 1536-well plates are "higher density" plates; 96-well plates are "lower density" plates), the use of white plates becomes more advantageous. This is due to the volumes used in these assay formats; assays run in 1536-well format are typically performed in 4-10 μL assay volumes. There is comparatively less fluorophore used in these assays, and maximizing signal may become more important as the amount of fluorophore in the well decreases. For more information, refer to our application note that compares black and white 96-well and 384-well plates in DELFIA TRF and LANCE TR-FRET assays.
Yellow DELFIA® plates
DELFIA yellow plates are semi-translucent yellow plates developed to have very low background autofluorescence in order to give optimal sensitivity in DELFIA TRF assays. In addition to providing increased sensitivity in many DELFIA assays, the DELFIA yellow plates are strongly recommended when running multiplexed DELFIA TRF assays (assays involving more than one DELFIA lanthanide - for example, Europium and Terbium chelates, or Europium and Samarium chelates, etc.)
DELFIA yellow plate
Microplates for cell-based fluorescent assays
The optimal 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:
- Do I need a sterile, tissue culture-treated plate?
- Does the plate need to be coated?
- 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:
- Strongly adherent cells
- Poorly adherent cells
- 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™ Adhesive Bottom Seal. BackSeal plate seals are available in either white or 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 plate wells.
| Plate | Description |
|---|---|
| CulturPlate™ |
|
| ProxiPlate™ |
|
| ViewPlate™ |
|
| CellCarrier™ |
|
Clear-bottom, TC-treated IsoPlate microplates could also be used for cell-based fluorescent assays. 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. IsoPlate microplates 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
- BackSeal plate seal can be used to convert a clear-bottom plate into an opaque (solid-colored) plate for top reads. BackSeal plate seal is offered in both white (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 plate wells.
Q. What kind of lid can I use for my plates?
A. Most CulturPlate, TC-treated ViewPlate, and ProxiPlate Plus TC plates are packaged with lids on the plates. Sterile lids can also be ordered separately (catalog number 6005619 for 96-well plates, catalog number 6007619 for 384-well and 1536-well plates). Lids should be removed prior to reading the plate to avoid damaging the plate reader. 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.
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 certain excitation or emission 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 fluorescent assays
Microplates for standard, in vitro fluorescent assays that do not require anchoring of cells or other reagents to the surface of the plate.
| Plate | Description |
|---|---|
| OptiPlate™ |
|
| ProxiPlate™ |
|
| ½ AreaPlate™ |
|
| ViewPlate™ (non-TC treated) |
|
| IsoPlate™ |
|
| VisiPlate™ (non-TC treated) |
|
Figure 3: Top view of a 384-well white ProxiPlate microplate (left) and view from underneath the same plate (right). The bottom of the wells is pushed towards the top surface of the plate to help increase signal while allowing use of low assay volumes.
Figure 4: 96-well OptiPlate microplate (left) vs. 96-well ½ AreaPlate microplate (right)
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.
*Clear-well 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. IsoPlate microplates 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). Additionally, IsoPlate microplates are only available in 96-well format, whereas ViewPlate microplates are available in 96-well, 384-well, and 1536-well formats.
Tips and FAQS- BackSeal plate seal can be used to convert a clear-bottom plate into an opaque (solid-colored) plate for top reads. BackSeal plate seal is offered (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 plate wells.
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 excitation or emission 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 OptiPlate and ProxiPlate microplates can be ordered separately (catalog number 6005617 for 96-well plates, catalog number 6007617 for 384-well or 1536-well plates). Lids should be removed prior to reading the plate to avoid damage to the plate reader. 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, which can lead to evaporation over longer periods of time.
Microplates for coated-plate assays (including DELFIA TRF immunoassays)
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.
| Plate | Description |
|---|---|
| OptiPlate™ HB (high-binding) |
|
| IsoPlate™ HB (high-binding) |
|
| DELFIA® uncoated plates |
|
| DELFIA® pre-coated plates |
|
- BackSeal plate seal can be used to convert a clear-bottom plate into an opaque (solid-colored) plate for top reads. BackSeal plate seal is offered (catalog number 6005199 for white, catalog number 6005189 for black). The color of the BackSeal plate seals should match the color of the sides of the plate well walls.
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 excitation or emission 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 OptiPlate and ProxiPlate microplates can be ordered separately (catalog number 6005617 for 96-well plates, catalog number 6007617 for 384-well or 1536-well plates). Lids should be removed prior to reading the plate. 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, which can lead to evaporation over time.
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:
- Antibody, protein, or sample (concentration of ~10 µg/mL or higher) is incubated in the plate overnight 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.
- Plate is washed three times with buffer (for example, 1X PBS).
- Plate is "blocked" overnight to cover the well surface area that remains (typically using BSA, sugars such as trehalose or casein, serum, etc.)
- Final washes are performed with buffer before using the plate in an assay.
See a detailed example plate coating protocol, given for a DELFIA immunoassay.
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
Application notes, posters and guides
Microplate catalog
Plate recommendations for 爱游戏平台注册登录 fluorescent assays
LANCE® TR-FRET assays
LANCE assays are TR-FRET assays using a Europium chelate as the donor fluorophore. Either white or black plates indicated for fluorescence applications can be used, though we typically recommend white plates for LANCE assays. LANCE assays utilize time-resolved fluorescence resonance energy transfer technology (TR-FRET) to generate a fluorescent signal at 665 nm. In contrast to prompt fluorescence assays, TR-FRET assays delay the signal acquisition for 50 microseconds in order to let any autofluorescent signal from the microplate or sample decay to background. 爱游戏平台注册登录 recommends white OptiPlate microplates for LANCE assays, both LANCE cAMP and LANCE Ultra kinase. Black microplates are generally used for prompt fluorescence assays to avoid the high autofluorescence generated by white plates. Black plates may also be preferred over white plates in situations where the assay generates a relatively high signal level that may cause well-to-well cross-talk in white plates. The disadvantage of using black plates is that a significant amount of the assay signal is absorbed by a black plate compared to a white plate. We have demonstrated the superior performance of white plates in LANCE assays by comparing:
- The performance of the LANCE Ultra cAMP assay in white and black 384-well OptiPlate microplates.
- In addition, we used a LANCE positive control to see if there is any issue with well-to-well cross-talk signal in both 1536- and 384-well white OptiPlate microplates.
Materials and Methods
Product Name | 爱游戏平台注册登录 Product Number |
|---|---|
OptiPlate-384 microplate, white | 6007290 |
OptiPlate-384 F microplate, black | 6007270 |
ProxiPlate-384 Plus microplate, white, shallow well | 6008280 |
OptiPlate-1536 microplate, white opaque | 6004290 |
LANCE Ultra cAMP kit | TRF0262 |
Standard cAMP curves were generated following the instructions in the LANCE Ultra cAMP kit. Samples were measured in triplicate in 384-well white and black OptiPlate microplates. The total assay volume was 20 µL per well. The data was analyzed using GraphPad Prism® software.
The LANCE positive control was used as directed to generate the signal for the cross-talk experiments. Wells not containing the LANCE control contained PBS. Cross-talk was measured in both adjacent and diagonal wells according to the platemap shown below.
Blank | Diagonal | Adjacent | Diagonal | Blank |
Blank | Adjacent | LANCE Control | Adjacent | Blank |
Blank | Diagonal | Adjacent | Diagonal | Blank |
The volume of LANCE control added was:
- 1536-well Optiplate microplate - 5 µL
- 384-well OptiPlate microplate - 20 µL
- 344-well ProxiPlate microplate - 10 µL
The volumes used are representative of typical assay volumes for these plate types.
Plates were read on the EnVision™ Multilabel Plate Reader using the following settings:
Light Source | Flash lamp |
|---|---|
Top Mirror | LANCE/DELFIA Dual |
Excitation Filter | UV (TRF) 320 |
Emission Filter | APC 665 |
Cycle | 200 |
Delay | 50 |
Number of flashes | 100 |
Results
The standard curves measured in the white and black 384-well OptiPlate microplates are plotted in Figure 1. Curve-fitting data is summarized in the table below the graph.
Figure. LANCE Ultra cAMP standard curves generated in white and black 384-well OptiPlate microplates.
The two standard curves gave similar EC50 and HillSlope values. The signal measured using a white plate was approximately 10-fold greater than that from the black plate, and the S/B ratio was also better using a white plate.
The well-to-well crosstalk measured in the various plate formats is shown in the table below.
| 384-well OptiPlate microplate | 384-well ProxiPlate microplate | 1536-well OptiPlate microplate |
|---|---|---|---|
Positive Control | 822,176 | 539,231 | 207,325 |
Blank | 496 | 477 | 790 |
Adjacent cross-talk | 461 | 502 | 837 |
Diagonal cross-talk | 469 | 464 | 829 |
Adjacent % cross-talk | 0.004 | 0.005 | 0.02 |
Diagonal % cross-talk | 0.003 | 0.002 | 0.02 |
Conclusions
White OptiPlate microplates are clearly superior to black OptiPlate microplates for performing LANCE assays. White plates give a much higher assay signal, and also a better S/B ratio than black plates. Autofluorescence from a white microplate is very low due to the time-resolved nature of the detection technology. Cross-talk in LANCE assays using white plates is not an issue, even in 1536-well format.
Recommended microplates for LANCE assays
White plates are recommended in all cases for performing LANCE assays. Depending on the desired well density and assay volume the following plates are suitable for LANCE assays:
- 384-well white Optiplate, CulturPlate and ProxiPlate microplates
- 1536-well white OptiPlate and CulturPlate microplates
- 96-well white OptiPlate, CulturPlate, ProxiPlate and ½-AreaPlate microplates
DELFIA® TRF assays
DELFIA assays are TRF assays using a Europium chelate, Terbium chelate, and/or Samarium chelate as the assay fluorophore. White, black, clear, or "DELFIA yellow" microplates are recommended for DELFIA assays. DELFIA yellow plates are semi-translucent yellow plates developed to give optimal sensitivity in DELFIA assays. DELFIA yellow plates are strongly recommended for multiplexed DELFIA assays.
The signal to background ratio obtained using a 96-well yellow DELFIA plate (catalog number AAAND-001) was compared to that of a clear 96-well SpectraPlate microplate (catalog number 6005640). A standard sample of 1 nM europium in DELFIA enhancement solution (in triplicate) was compared to a background sample of wells filled with PBS. The results shown in the table below demonstrate that the low background signal generated by the yellow DELFIA plates leads to a much higher S/B ratio.
| Yellow DELFIA plate | Clear SpectraPlate microplate |
|---|---|---|
Average Signal | 266,011 | 242,130 |
Average Background | 135 | 871 |
S/B Ratio | 1,975 | 278 |
Plate seals
爱游戏平台注册登录 offers a variety of plate seals. TopSeal plate seals are applied to the top surface of the plate, and are generally used to prevent evaporation or contamination during assay incubation steps and/or plate reading measurements. Note that TopSeal plate seal has spectral properties that may interfere with an assay measurement; you may need to remove the seal prior to reading your plate. BackSeal plate seals are applied to the bottom of the plate. BackSeal plate seals can be used to change a clear-bottom plate into a white- or black-bottom plate in order to reduce cross-talk during top-reading measurements.
Table. Plate seal products.
| Product | Type of seal | Plate format | Number of seals | Catalog number |
|---|---|---|---|---|
| TopSeal-A plate seal | Clear adhesive seal | Any | 100 | 6050185 |
| BackSeal plate seal | White adhesive seal | (all) | 55 | 6005199 |
| Black adhesive seal | (all) | 55 | 6005189 |
Custom plate services at 爱游戏平台注册登录
爱游戏平台注册登录 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:
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