Microplates for Radiometric Assays

Overview

Assays involving radiochemicals can be run in various medium- and high-throughput plate-based formats, including filtration assays (commonly used for thymidine incorporation and radioligand binding assays), liquid scintillation counting assays, scintillant coated-plate assays (commonly used for HPLC fraction analysis or  ⁵¹Cr release assays), and proximity assays (using SPA beads or Cytostar-T™ plates). Plate recommendations for each assay format are detailed in the sections below. Radiometric assays that measure signal coming from a beta-emitting radioisoe (such as ³H, ¹⁴C,  ³²P, and³⁵S) or a radioisoe that releases Auger electrons (such as ¹²⁵I and ⁵¹Cr) typically rely on addition of a scintillant, which captures the energy from the emitted beta particle and, in turn, releases energy in the form of photons that can be measured on radiometric instruments (such as the Revvity MicroBeta or Count Plate Counters) that use a photomultiplier tube for detection. Because of this, plate selection for these assays will factor in some of the same considerations as for standard luminescence assays. Liquid scintillation counting assays require the use of liquid scintillation cocktails, which can sometimes contain harsh organic solvents. For these assays, compatibility of the plate material with the cocktail solvent should be a consideration. Additional considerations such as whether the detection instrument is reading from the  and/or bottom of the plate should also be factored in when choosing a plate. Clear-bottom plates allow for bottom-reading and coincidence measurements on instruments such as the MicroBeta plate counter. Clear-bottom plates with black or white wells can be used in conjunction with BackSeal™ Adhesive Bottom Seal to reduce cross-talk in -reading measurements. Solid opaque plates need to be measured with -reading instruments, such as the Count system (the MicroBeta counter can also support -reading measurements). 

Plate color

Signal

The use of white plates will result in higher signal for photon measurement assays (for example, radiometric assays that use a scintillant to convert energy from a beta-emitting or Auger electron-emitting radioisoe into photons), whereas black plates can "quench" the signal by absorbing some of the light produced by the assay. For most radiometric applications, we recommend the use of white plates, or white-walled plates with clear bottoms for bottom-reading instruments. However, if you are working with an assay that gives a strong signal, black or gray plates may be helpful in reducing background from phosphorescence (see next section). 

Phosphorescence and dark adaptation

For assays that involve use of a scintillation cocktail or other scintillant (SPA bead, Cytostar-T plate, LumaPlate™, MeltiLex™), phosphorescence may be a potential issue. Radiometric assays that measure signal coming from a beta-emitting radioisoe (such as 3H, 14C, 32P, and 35S) typically rely on addition of a scintillant, which captures the energy from the emitted beta particle and, in turn, releases energy in the form of photons that can be measured on radiometric instruments that use a photomultiplier tube for detection. Assays involving radioisoes that emit Auger electrons (such as ¹²⁵I and  ⁵¹Cr) can also use scintillants in a similar manner.

Background phosphorescence is a consideration when choosing between white or black plates for light-producing (photon-producing) assays. Phosphorescence is the emission of light by a substance, resulting from stored energy. Certain components in a buffer or sample may phosphoresce, and plastic from the microplate itself can also phosphoresce. Phosphorescence can lead to increased background, which could potentially have a negative impact on a given assay. Black or gray microplates intrinsically exhibit less phosphorescence than white microplates, and may be desirable for particular assays. Alternatively, assays run in white microplates can be "dark-adapted" by shielding the microplate from light prior to reading the plate in order to reduce background phosphorescence. You can start by dark-adapting the plate for 5 minutes. The amount of time required for dark adaptation will vary from assay-to-assay, and should be optimized.  

Cross-talk

There are two types of cross-talk: optical and isoic (or radiometric) cross-talk. Optical cross-talk occurs when light from one well travels to another well where it is detected and adds non-specific counts to that well. Isoic cross-talk occurs when high energy beta particles from one well travel to adjacent wells where they interact with the scintillant in that well, adding to non-specific counts. This can be significant when high energy isoes are used, such as ³²P.

Microplates recommended for use in radiometric assays are thick-walled rigid plates containing colorants which produce an opaque white, black, or gray finish. These pigments, in conjunction with wall thickness, help attenuate the transmission of radioactive decay energy between neighboring wells, which would otherwise lead to "cross-talk". The use of opaque (solid-color) microplates can also reduce optical cross-talk to negligible levels. 
 

Microplates for filtration assays

We offer glass fiber UniFilter™ plates (GF/B and GF/C) for filtration assays. The difference between GF/B and GF/C filters is the filter thickness (GF/B is 680 µm and GF/C is 260 µm), resulting in a different pore size (1 µm or 1.2 µm, respectively). The size of the pore determines how much sample is retained on the filter, and how much is filtered as a waste material. Assays involving cell membrane preparations can be run using either GF/B or GF/C filters. For whole cell-based assays, we recommend GF/C filters to prevent clogging.

UniFilter plates are also available with a polyethyleneimine (PEI) coating. PEI is a cationic polymer that can neutralize the negative charge of the glass fiber filter, and can be used when a positively-charged ligand or other reagent exhibits high non-specific binding to the glass fiber filter.

UniFilter plates should be used in conjunction with BackSeal (catalog number 6005199 for white, catalog number 6005189 for black) and Seal™-A adhesive seals. BackSeal adhesive seal fits onto the bottom of the filter plate, which should be applied before addition of scintillation cocktail. Seal-A adhesive seal fits on of the plate to prevent spills/contamination when placing the plates into the high-throughput detection instrument.

Both the Revvity Count and MicroBeta plate counters can be used to measure filtration assays. 

Table 1: Filter plate products

*The Harvest Plate is a 96-well filter plate designed for use with the Tomtec™ Mach IIIW automatic harvester. The Harvest Plate includes a retaining plate that is designed to isolate each of the 96 filter areas and also to support the filter material. The Harvest Plate is made up with GF/C glass fiber. Other filter types are available on request. The external dimensions of the Harvest Plate are identical to a standard 96-well microplate and it is therefore highly suitable for use in automated systems.

Alternatively, we offer filtermats that can be placed into cassettes compatible with the high-throughput detection instrument being used (Table 2) or filtermats that are used with the FilterMate Universal Harvester or in the OmniFilter™ Plate Assembly (Table 3). 

Table 2. Filtermat products (to be used with cassette to adapt to high-throughput detector) 

 

Table 3. Filtermat products to be used with FilterMate Harvester or OmniFilter plate to adapt to high-throughput detector 

Microplates for liquid scintillation counting assays

Liquid scintillation counting assays require the use of liquid scintillation cocktails, which can sometimes contain harsh organic solvents. For these assays, compatibility of the plate material with the cocktail solvent should be a consideration. PicoPlates are composed of solvent-resistant Barex® plastic. OptiPlates, Isoplates, and VisiPlates are composed of polystyrene, and are less suitable for use with liquid scintillation cocktails that contain harsher solvents. The Count and MicroBeta instruments from Revvity can be used to measure liquid scintillation assays. 

OptiPlate™ microplates 

OptiPlate microplates are standard, highly-versatile polystyrene microplates. White or black OptiPlate microplates can be used in radiometric detection assays. OptiPlate microplates are solid (opaque) in color with no transparency, and therefore require measurement in -reading microplate readers. OptiPlate microplates are available in 24-well, 96-well, 384-well and 1536-well formats.

Isoplate™ microplates (untreated) 

Clear-well Isoplate microplates are polystyrene plates that have a clear bottom, while the sides of each well are either black or white. This makes clear-well Isoplate microplates suitable for bottom-reading instruments or coincidence measurements. 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. Clear-well Isoplate microplates were developed for coincidence counting in a MicroBeta instrument (reading from  and bottom coincidentally). Isoplates are only available in 96-well format.

VisiPlate™ microplates (non-TC treated) 

VisiPlate microplates are polystyrene plates that have a clear-bottom base, with the sides of each well a solid white or black 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). VisiPlate microplates are similar to Isoplate microplates, but are our only clear-bottom, solid-colored well plates that are offered in 24-well format. VisiPlate microplates are only offered in 24-well format.

Flexible PET plates

Flexible plates are designed for Revvity's MicroBeta liquid scintillation counter, and give the best results in liquid scintillation counting and SPA assays. The plates are made of clear polyethylene-A (PET-A) or polyethylene-G (PET-G) plastic and printed with grid lines between wells to prevent cross-talk. The plate is chemically resistant to all DIN (di-isopropyl naphthalene)-based cocktails and is suited for chromium release, Cerenkov ³²P, SPA and all other general liquid scintillation applications. Plate cassettes and support frames are required to use the flexible microplates in the MicroBeta instrument. Flexible PET plates are available in 24-well and 96-well format.

LumaPlate™ microplates

The LumaPlate is a solid scintillator-coated microplate that eliminates the need for adding liquid scintillation counting cocktail. Scintillant is coated onto the bottom and sides of wells. This can offer ease of use. LumaPlate is recommended in use of HPLC or ultraPLC fraction analysis in metabolic studies and 51Cr release assays. LumaPlates are microplates that come in 96-well (shallow or deep-well) and 384-deep-well format. 
 

Microplates for SPA assays

We recommend the use of white plates or white-walled, clear bottom plates for SPA bead-based assays.

OptiPlate™ 

OptiPlates are standard, highly-versatile polystyrene microplates. They are solid (opaque) in color with no transparency, and therefore require measurement in -reading microplate readers. OptiPlate microplates are available in 24-well, 96-well, 384-well and 1536-well formats.

ProxiPlate™
 
ProxiPlate microplates are shallow-well plates designed for low-volume assays. The bottom of the wells is raised to position the surface of the liquid in each well as close to the -reading instrument detector as possible, resulting in high signal. They are solid (opaque) in color with no transparency, and therefore require measurement in -reading microplate readers. ProxiPlate microplates are available in 96-well and 384-well formats only.

Isoplates (untreated) 

Clear-well Isoplate microplates are polystyrene plates that have a clear bottom, while the sides of each well are either black or white. This makes clear-well Isoplate microplates suitable for bottom-reading instruments or coincidence measurements. 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. Clear-well Isoplate microplates were developed for coincidence counting in a MicroBeta instrument (reading from  and bottom coincidentally). Isoplate microplates are only available in 96-well format.

VisiPlate™ 

VisiPlate microplates are polystyrene plates that have a clear-bottom base, with the sides of each well a solid white or black 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). VisiPlate microplates are similar to Isoplate microplates, but are our only clear-bottom, solid-colored well plates that are offered in 24-well format. VisiPlate microplates are only offered in 24-well format. 

ScintiPlate^™ and Cytostar-T plates for proximity assays

ScintiPlate, and Cytostar-T plates are coated with scintillant, and therefore do not require the addition of scintillation cocktail for your assay. 

ScintiPlate^™ microplates

ScintiPlate microplates are clear-bottom plates with white walls, but are otherwise similar to FlashPlate microplates, in that they are special plates that contain scintillant embedded into the plastic of the plate and are intended for use in solid-phase (coated-plate) assays. In a ScintiPlate format, some component of the assay is captured onto the bottom and sides of the well of a plate. The assay is designed so that when radiochemical binds or is utilized as a substrate, this puts the radiochemical into close proximity of the bottom and sides of the ScintiPlate microplate. The beta-energy from the radiochemical interacts with scintillant embedded in the plastic of the well, producing signal. Unbound or unused radiochemical will be floating freely in solution, and will not be close enough to the edges of the well to produce signal. Because this is a proximity assay, no wash steps are required (though you can wash if desired).

ScintiPlate microplates are offered with streptavidin-coating, tissue culture-treated (for use in cell-based assays), or uncoated for direct coating procedures. Because they have clear bottom wells, they can be used for bottom-reading or coincidence measurements. Common applications include:

• IRMA sandwich assays - e.g. T4 competitive immunoassay
• RIA competitive assays - e.g. estradiol
• Coated receptor assays - e.g. cloned G-protein or fast ion-receptor assays
• Hybridization assays - e.g. DNA/RNA hybridization or detection of point mutations
• Enzyme activity assays - e.g. kinase with biotinylated substrate
• Cell binding studies - e.g. 2-site antibody to cell-surface receptors
• Binding experiments involving a labelled biotinylated compound - e.g. biotinylated substrate
• Kinetic assays involving a tritiated compound - e.g. an antibody  

Cytostar-T plates

Cytostar-T scintillating microplates are sterile, tissue culture treated microplates designed not only for the growth of adherent but also suspension cell cultures. The integral, planar, transparent base of each well is composed of a proprietary homogeneous mixture of scintillants and polystyrene. The transparent nature of the base permits the observation of growth of cells plated in the well. Radioisoes having suitable decay characteristics (3H, 14C, 35S, 45Ca, 125I) brought into proximity with the scintillant contained within the base (via radiochemical uptake or radiochemical interaction with the cells) will have that radioactive decay converted to a light signal. The amount of light generated is proportional to the amount of radioisoe within, or associated with, the cells. 

Choosing a scintillating plate