This guide provides important information to help you make the right decision. Learn about the different types of readers/detectors; read modes, applications, technologies and other important considerations.
Microplate readers are used to detect biological, chemical or physical events in microtiter plates via the measurement of light. Microplate readers/detectors are the key workhorses in many laboratories and are used extensively for many applications across a wide range of disciplines including life sciences, drug discovery, bioassay validation, quality control, drug safety, toxicity testing, clinical diagnostics and biopharmaceutical/pharmaceutical manufacturing processes. Although a well-established product category, microplate readers continue to evolve towards greater functionality, flexibility, speed and throughput. Currently, there are a wide variety of microplate readers available, offering different capabilities and functionalities.
Microplate readers differ by the type of detection mode. The most common detection modes are absorbance, fluorescence and luminescence. Additional detection modes include fluorescence resonance energy transfer (FRET), time-resolved fluorescence (TRF), fluorescence polarization, bioluminescence resonance energy transfer (BRET), AlphaScreen and nephelometry.
Microplate readers also differ by detector technology. Light detection can be performed using filters, monochromators or spectrophotometry. Monochromator-based technology has the most flexibility in wavelength detection, so is most suitable for labs with variable detection requirements. Newer spectrometer-based readers can capture an entire absorbance spectrum of wavelengths, offering even more flexibility in detection. Filters are for fixed wavelength detection but offer greater sensitivity, so are more appropriate for labs with limited detection needs. Hybrid systems combining these technologies are also available. In addition, lasers rather than flash lamps are better for time-resolved fluorescence and AlphaScreen detection.
Microplate readers can be purchased as either single-mode or multimode; the latter combines several read modes in one instrument. Multimode microplate readers enable researchers to perform multiple assay types in one system. Multimode readers are typically more expensive than single-mode readers but purchasing one multimode reader is likely to be more cost-effective than buying several dedicated machines. In addition, they do not require much more bench space than a single reader and are often modular and upgradable, enabling a laboratory to purchase only what they need at the time but allowing for future additions.
For example, the CLARIOstar® microplate reader by BMG LABTECH is a multi-mode reader equipped with a patented new, dual monochromator technology, named LVF (linear variable filter) MonochromatorTM technology, offering fluorescence and luminescence measurements. The linear variable filters in the CLARIOstar® have variable coatings along their lengths that can reject or pass certain wavelengths of light. With no stray light and fewer background signals, the LVF Monochromator technology provides a higher-sensitivity detection.
Plus, for measuring luminescence assays, it is important to ensure that the dynamic range offered by the microplate reader suffices the variety of experiments that will be planned in the future. The CLARIOstar®, for example, offers a nine-log dynamic range, providing you with flexibility in experiments and ensuring no signal goes undetected.
When choosing a microplate reader/detector, the first, and perhaps the most important, consideration is the application requirement. Microplate readers are used across diverse scientific disciplines for varying applications. The most common methods are briefly introduced below. This downloadable compendium of application notes produced by BMG LABTECH covers a wide range of applications in the field of life sciences, spanning from bacterial kinetics, to live-cell assays to cell signaling measurements.
Protein quantification is a common application where microplate readers are employed. Protein concentration is measured using chromogenic assays by determining absorbance at 280 nm. Microplate readers provide a way to scale up the number of samples processed in protein quantification, while also enabling triplicates in the same experimental conditions.
This downloadable application note by BMG LABTECH provides you with predefined protocols in the CLARIOstar® microplate reader that you can use for protein quantification experiments.
Plus, delve deeper into using microplates in four commonly used colorimetric protein assays and explore a fluorescence-based protein quantification using microplates.
Enzyme-Linked Immunosorbent Assays (ELISAs)
ELISA is an immunodetection assay that uses labeled secondary antibodies to detect specific antigens. The secondary antibody conjugate, such as horseradish peroxidase (HRP), is detected via an enzyme-mediated chromogenic change. ELISAs are used extensively in the life sciences, pre-clinical research and clinical diagnostics. The majority of plate readers are able to read ELISAs.
Nucleic acid quantification
DNA and RNA absorb light within the UV range. Absorbance of a sample at 260 nm can be used to calculate the concentration of double-stranded DNA, single-stranded DNA or single-stranded RNA. Protein contamination, common in DNA and RNA preparations, can be determined using the 260/280 nm absorbance ratio. Plate readers with UV bandwidth detection capabilities are suitable for this detection.
In recent times, with an increased interest in sample conservation and high-throughput analyses, microplate readers are more preferred over the traditional cuvette-based measurements. This application note details the use of microplates in analyzing smaller volumes, taking a step towards automation.
Reporter genes, such as luciferase or GFP, can also be assessed in microplate readers, enabling in vitro and in vivo determination of gene expression for studies using markers of genetic alteration. Any reader with appropriate wavelength detectors can be used for this detection.
This application note by BMG LABTECH outlines how CRISPR/Cas9 genome editing was used to endogenously express nanoBRET donor proteins, enabling measurements of protein interactions and trafficking.
Protein-protein and protein-ligand interactions
Using BRET, it is now possible to measure protein-ligand or protein-protein interactions in a microwell format. The first use of BRET for monitoring ligand binding to GPCRs in live cells is outlined in this useful application note by BMG LABTECH. The CLARIOstar microplate reader enabled calculation of IC50 and KD values from saturation and competition binding assays.
Plus, learn key elements of measuring biomolecular interactions using Bio-Layer Interferometry in this application note that discusses large molecule kinetic assays on the Octet platform by Pall Life Sciences – ForteBio.
Measurement of enzyme activity is a common assay in life sciences and pre-clinical research. The time-resolved enzyme-dependent accumulation of a marker or product can be used to determine enzyme kinetics. A majority of plate readers have this capability.
Microbiologist, Dr. Jason Sylvan, Assistant Professor of Oceanography, Texas A&M University, who samples microbes from the depths of ocean, measures microbial enzyme activity using the portable Spark® microplate reader by Tecan that he carries with him on the ship. Read the full article here.
Fluorescent markers such as EGFP, YFP, mCherry and mTomato or fluorescent biosensors can be used to measure a variety of real-time cell-based activities, including, intracellular transport, protein signaling, receptor desensitization, migration, division, apoptosis, metabolism, differentiation, chemotaxis, transcription and translation.
In an exclusive interview with SelectScience, the founder of Montana Molecular, Anne Marie Quinn, shares how her team measures, in real time, multiple GPCR signaling pathways inside a living cell using the CLARIOstar microplate reader.
Fluorescent or luminescent markers are also used in pharmacological and drug discovery assays. These are used to investigate ligand binding and protein interactions. TTP Labtech’s ameon® lifetime reader incorporates fluorescence lifetime technology, enabling cost-effective screening of common drug targets as well as challenging epigenetic targets. Read about a novel nanoBRET assay developed in association with BMG LABTECH. In this SelectScience video, Dr. Kevin Pfleger, Associate Professor, University of Western Australia, describes how he uses nanoBRET technology along with the CLARIOstar and PHERAstar FSX microplate readers to develop high-throughput screening assays for developing GPCR-targeted drugs.
Bacterial cell counting can be determined by optical density at 600 nm, which indicates the growth phase for harvesting the cells. To determine confluence of basic cell culture, many modern microplate readers are equipped with a ‘confluence count’ setting. For example, this downloadable method compares the cell counting feature of the Spark multimode microplate reader with a GFP-labeled cell count.
The CLARIOstar microplate reader by BMG LABTECH is equipped with an advanced cell layer scanning capability. This enables up to 900 points to be measured in each well, a feature perfect for unevenly distributed cells.
There are many different commercially available cell-based assay kits that enable cell viability, proliferation and apoptosis to be measured. These include those for monitoring ATP, measuring caspase activity and detecting bromodeoxyuridine (BrdU). They are also used in toxicity testing in pre-clinical drug development.
These assays, applied to live cells, require a microplate reader to maintain appropriate environmental controls. BMG LABTECH’s CLARIOstar® comes with a module – the Atmospheric Control Unit (ACU) – for independent control of oxygen and carbon dioxide.
Read about the monitoring of apoptosis and necrosis in real time using the CLARIOstar’s ACU feature, that maintains the appropriate O2 and CO2 environment for cells, while the RealTime-GloTM Annexin V Apoptosis and Necrosis Assay measures cell death using luminescence and fluorescent signals.
In live, real-time cell-based experiments, it is beneficial to read from the bottom of the microplate, rather than the top. Reading from the bottom offers several advantages for cell-based detection: the light collector can be placed closer to the sample and the cell layer adherent closer to the bottom of the well, which decreases light dissipation. Moreover, the interfering effect of the cell culture medium is significantly reduced. Both factors positively affect sensitivity. In addition, bottom reading allows for a cover or lid to be placed on top of the microplate to prevent cell contamination and liquid evaporation. This is particularly important in time-lapse experiments.
Several readers are optimized for live-cell assays, including BMG LABTECH’s PHERAstar® FSX and CLARIOstar® readers, which use a proprietary Direct Optic Reading system to eliminate the need for fiber optics.
Live-cell experiments involving translational research concepts, such as testing for ischemia-reperfusion in cells for research on stroke, involve a tight regulation of oxygenation as measurements are made in real time. Read an example of such a study using the ACU to control for oxygenation levels mimicking conditions of a stroke in vitro.
Cell migration in wound healing assays is a popular application using microplate readers. Learn how you can use a microplate reader to automatize the Radius 96-well Cell Migration Assay.
Recently, there has been an increased drive to develop assays and readers for 3D cell cultures. These spheroids or cell clumps can be grown in multi-well plates and more closely mimic the endogenous environment, which is important in both life sciences and drug discovery applications. Watch the video with Dr. Kirk McManus, Associate Professor, University of Manitoba, and Senior Scientist, CancerCare Manitoba, to learn how temporal resolution of 3D tumor spheroids in colorectal cancer is obtained using the Cytation microplate reader by BioTek.
In another method, 3D heart muscle was reconstituted with cardiac myocytes derived from human induced pluripotent stem cells (iPSCs). Learn how calcium flux is measured every 0.01 seconds using the CLARIOstar®.
Phenotypic screening is used routinely in drug discovery for the identification of substances that alter the phenotype of a cell or an organism. The target cells are screened with compounds or biopharmaceuticals to assess modulation of the activity of interest. In vitro phenotypic screening uses cell lines and cell-based assays and is often performed in high-content microplates and may utilize live cells.
Deciphering cell signaling, especially for G-protein coupled receptors (GPCRs), forms the basis of discovering therapeutic targets. In fact, a predominant number of current drugs target the GPCR signaling. Upon ligand binding, the Gs-coupled GPCR receptor activates adenylyl cyclase that in turn produces cAMP, governing important cellular responses. Gi-coupled GPCR receptors, however, inhibit adenylyl cyclase and cAMP production. The phosphatidylinositol pathway, on the other hand, is triggered by the Gq-coupled receptors. In Gq-coupled signaling, DAG and intracellular calcium act as second messengers, ultimately influencing cell functions.
Microplate readers can be used to detect GPCR signal transduction in living cells. Two methods published by BMG LABTECH describe (1) real-time detection of Gs and Gi signaling and (2) detection of GPCR second messengers in living cells.
Drug discovery and manufacturing applications often require high-content screening (HCS) and/or high-throughput screening (HTS). Most of the applications above can be conducted in high-content, multi-well plates in microplate readers. High-throughput drug screening for target validation and ADMET (absorption, distribution, metabolism, elimination and toxicity) is a key process in drug discovery. However, phenotypic cell-based assays are now becoming more commonplace. In addition to conventional assays, these readers enable phenotypic screening of cells, either by well or per cell. Multimode microplate readers have been designed specifically for this HTS combined with imaging.
Amplified Luminescent Proximity Homogeneous Assay Screen (AlphaScreen)
AlphaScreen is a versatile assay technology developed to measure analytes using a homogenous protocol that enables sensitive and precise interrogation of signaling pathways, receptors, enzymes and kinase targets, in a cell-based format. AlphaScreen has been particularly useful for screening GPCRs, growth factor receptors, intracellular MAPK inhibitors and many other signaling pathways.
Homogeneous Time-Resolved Fluorescence (HTRF)
Utilizing rare-earth lanthanides with long emission half-lives as donor fluorophores, HTRF technology combines standard FRET with the time-resolved measurement (TR) of fluorescence. HTRF is commonly used for GPCR and kinase screening, two of the most important target classes investigated within drug discovery. Other HTRF applications include discovery of new biomarkers, studies of protein-protein interactions, epigenetics and an alternative method for bioprocess monitoring.
Using the PHERAstar® FSX microplate reader by BMG LABTECH, the HTRF IP-One assay was performed where single clones were functionally screened using stably transfected recombinant CHO-M1 cells. Download the full method here.
Microplate readers/detectors are used for numerous applications and, as technologies evolve, even more will emerge. When purchasing a microplate reader/detector, it is important to establish what applications you will be using it for now, and in the future. You will want to ensure that your chosen microplate reader/detector is capable of fulfilling all your requirements but also consider how easily upgradable it is for your future needs.
Request a demo: Once you have considered both your application needs and the different technologies that are available, chances are that you will have narrowed down the choice to just a few microplate readers. Before going ahead and purchasing a microplate reader/detector, it is highly recommended that you try it out. Most manufacturers will let you demo the instrument for a trial period. This is a great opportunity to ensure the microplate reader fulfills all your requirements, is easy to use and to make sure you get the right support and training to use the instrument.
Technical support and warranty: Consider the extent of the technical support and training that is available from the manufacturer at the point on purchase. Is it included in the purchase cost? It is also recommended that you look into the company’s policy on after-sales service and standard warranty. While everything is operating well, this may be the last thing on your mind, but in the event that after-sales support is required, it is important to know how accessible that is.
Accessories and add-ons: Not all microplate readers have the same standard features. Be sure to ask what is included with the reader at the time of demonstration. Optional items may be key parts of the kit, including computers to run the instrument, filters or filter cubes, software licenses, software upgrades, FDA-compliant software, environmental controls or injectors.
Software: Microplate readers take hundreds or thousands of measurements, so data acquisition and analysis are key to efficient workflow. Make sure that you know how to use the software for your current needs and how to keep it updated. Find out how well it is supported by the manufacturer.
Compatibility: A lab can have equipment from different manufacturers. Consider how well your chosen reader will integrate with your existing equipment and how easy it will be to upgrade in the future. Many models have a modular design and add-ons, but their compatibility may be brand limited.
Future needs: Taking current and future trends into consideration is essential in maximizing the lifespan of the microplate reader/detector. As systems become more sophisticated, integration, convenience and application specificity will be fundamental. Systems will become faster and more efficient and be able to run a larger variety of different assays. Application possibilities will increase as manufacturers work to provide solutions to individual and industry demands. Manufacturers are taking customer feedback and requirements into consideration, with many developing strategies for even greater specificity assays and technology for the future.
Whether purchasing a microplate reader/detector for a new application or replacing an existing system, there are a number of factors to consider. You will need to examine your current and future application needs and determine which of the available technologies best suits these applications. Visit the SelectScience Microplate Readers / Detectors product directory for an overview of the latest products from leading manufacturers. Keep up-to-date with the latest techniques and advances in technology by visiting the SelectScience Microplate Readers / Detectors page in our life sciences community for interviews, application notes, videos and the latest news.