SPECTROstar Nano
Absorbance plate reader with cuvette port
When scientists carry out experiments, they often use technologies and assays to make critical measurements in the laboratory. Accuracy is crucial but there is also often a need to make as many robust measurements as possible in the shortest possible timeframe. Microplate readers allow researchers to perform many different types of assays in a way that is consistent with these requirements for automation and performance.
Broadly speaking, biological assays fall into two categories when measurements and time are considered.
In the first group, endpoint assays require making a single measurement at a selected point in time. Kinetic assays, also known as continuous assays, are a second category and used for multiple measurements over time, allowing real-time monitoring of reactions.
Kinetic assays may be performed on a microplate reader1 either in well mode (fast kinetics) or plate mode (slow kinetics). This is the terminology used in BMG LABTECH’s MARS software. Users of microplate readers may encounter different terminology for well and plate mode depending on the reader and its accompanying software but the principles remain the same (for example, well mode is sometimes referred to as well wise in some software). The main difference between endpoint and kinetic (continuous) modes is therefore that endpoint mode measures at a single time point, while kinetic or continuous measurement protocols track the reaction progress continuously until completion.
Endpoint and kinetic mode assays are widely used by researchers in the life sciences. They find many applications including for example studying reaction rates and the mechanisms of biological reactions, performing Enzyme-Linked Immunosorbent Assays (ELISAs), making growth curve measurements, and performing high-throughput screening assays for drug discovery.
In this blog, we look at the different types of endpoint and kinetic mode assays, provide examples of applications for each, and look at some of the features that facilitate using microplate readers for endpoint and kinetic mode measurements.
What are the differences between endpoint and kinetic modes on a microplate reader?
Endpoint mode, plate mode and well mode are the different settings on a microplate reader used to measure endpoint and kinetic mode assays. The main difference between endpoint and kinetic modes is that endpoint mode involves a single measurement per well after a specified time period, providing information about the final state of a reaction, while kinetic modes involve multiple measurements over a defined time period to monitor changes in the reaction. Endpoint mode is simple and fast, making it suitable for large-scale studies and routine tests. High-throughput screening often prioritizes speed and low cost over detailed mechanistic information, making endpoint mode favorable.
For the different kinetic modes of a microplate reader, plate mode and well mode are used to take multiple measurements over time and are suitable for slow and fast kinetic measurements, respectively. In plate mode, each well is measured once and the measurements for each well are then repeated multiple times over a defined time period. In well mode, different measurements are made over time in the same well (up to 100 per second and then the reader moves onto the next well for the next series of kinetic measurements. Kinetic mode, for example, is critical for later stages of drug discovery such as lead optimization and mechanism determination. Beyond drug discovery, kinetic mode finds use in many assays in the life science laboratory that require monitoring reactions over time. Kinetic mode provides a more detailed view of the entire process from start to finish compared to endpoint mode.
Assays in endpoint and kinetic modes
Endpoint assays and the different kinetic modes of a microplate reader collectively cover many of the fundamental assays performed in the life science laboratory. Endpoint and kinetic mode assays are commonly used to measure the concentration of an analyte in biological samples. It is essential to follow a standardized procedure in these types of assays, including steps like reagent dispensation, incubation, and measurement, to ensure accurate and reliable results. A microplate reader delivers excellent performance for standardized procedures and also offers the flexibility to enhance for future requirements.
Examples of endpoint assays include ELISA assays, DNA and protein concentration determination, steady-state dose response curves, and assays like ADP-Glo™ or Transcreener™ GDP fluorescent polarization assays, where the measurement of total analyte concentration or total absorbance is a key outcome.
Examples of well mode fast kinetic assays include calcium flux measurements, enzyme kinetic studies, ion channel activity measurements, transcription assays, and dual luciferase reporter assays (DLR). Essentially any assay with rapid changes of signal over time warrants measurement on a microplate reader in well mode.
Slower kinetic assays suitable for plate mode include measurement of bacterial and yeast growth, cell migration and invasion assays, enzyme kinetic assays, and oxygen radical absorbance capacity or ORAC assays. These are reactions where kinetic changes are taking place on a slower timescale. Here measurements can be effectively made by successive evaluations in a well that can accommodate detection in each well of a microplate before the second measurement commences.
Here we give some examples of applications that can be performed on BMG LABTECH microplate readers in endpoint and kinetic modes.
Endpoint assays
Endpoint assays are easier to perform and generally require fewer resources than kinetic assays. In endpoint assays, reagents or samples are added at specific steps according to the protocol, and the reaction is allowed to proceed until completion. The measurement is then taken after the reaction has fully progressed, ensuring accurate assessment of the final product or substrate concentration.
ELISA assays
ELISAs are used to detect or measure specific biological molecules (analytes) in complex mixtures of biomolecules and find many applications in diagnostics, drug discovery or fundamental research. Colorimetric or absorbance assays are widely used for ELISA assays and horseradish peroxidase and alkaline phosphatase are the two most encountered bioconjugated enzymes used in these colorimetric assays. The readouts for these assays are endpoint measurements at specific wavelengths. To ensure accurate quantification of the analyte, calibrators with known concentration are used to generate a calibration curve, which is essential for calculating the concentration of unknown samples. Calibration is crucial in ELISA assays to maintain measurement accuracy and reliability.
In the application note Overview of ELISA assays and NADH/NADPH conversion detection different examples of endpoint assays are given for the use of these enzymes and substrates in measuring classic ELISA assays.
Protein quantification
Researchers routinely need to measure the concentrations of proteins and DNA in biological samples. Assays like Bradford, Lowry and the bicinchoninic acid (BCA) assay are some of the most frequently encountered methods for the determination of protein concentrations. In these assays, the amount of product formed from the reaction is measured, and this product is proportional to the protein concentration. Each depends on an endpoint absorbance measurement at a specific wavelength, which detects the product generated during the assay. For the Bradford assay, for example, samples are read at 595 nm. The application note Absorbance-based methods for protein quantification on BMG LABTECH Instruments describes the use of these different endpoint assays for protein determination and you can read more widely about protein quantification in the blog "Protein Assays".
DNA quantification
DNA quantification is possible using absorbance- and fluorescence-based methods and many of these assays are performed as endpoint assays. In the application note DNA quantification using absorbance (A260) and fluorescent methods (QubitTM and Quant-iTTM/PicoGreenTM) examples are presented of some of the commonly used methods for DNA quantification based on endpoint fluorescence and absorbance methods (Fig. 1). Each of these methods can be used to quantify DNA at different concentrations, sample volumes, and at the required throughput on a microplate reader.
Transcreener® assays
Transcreener® assays are high throughput tools that can be used to study thousands of targeted enzymes due to their ability to measure the accumulation of reaction products such as ADP, AMP/GMP, UDP and GDP nucleotides. Transcreener assays measure the amount of product formed during the reaction to determine enzyme activity or analyte concentration. They are typically endpoint assays since measurements are made after binding equilibrium is established and the reaction between nucleotide and the tracer antibody has stopped.
In the application note Methyltransferase, acetyltransferase, kinases, and GTPases can all be measured with Transcreener assays examples are presented of how the PHERAstar® FSX can be used to make fluorescence, fluorescence polarization, and Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) for different Transcreener assays including those used for epigenetic studies of methyltransferase enzymes.
Transcreener assays can also be used to obtain steady-state dose-response curves from endpoint measurements obtained at different concentrations of inhibitors. These types of measurements allow half-maximal inhibitory concentrations (IC50) or half maximal effective concentrations (EC50) values to be obtained (Fig. 2). They are suitable for studies of kinase inhibitors, for example, with different fluorescent readouts. Some examples of these types of measurements are given in the application note Transcreener ADP2 FP assay certification for BMG LABTECH instrumentation.
ADP-GloTM Kinase Assays
The ADP-GloTM Kinase Assay is another example of an assay that is typically used to measure endpoints. In this type of assay, a kinase reaction is run for a fixed time and ATP is converted to ADP. At specific steps in the protocol, reagents are added, such as when the reaction is stopped by adding ADP-Glo reagent which depletes the remaining ATP. A second reagent is then added to convert ADP to ATP, and this ATP drives a luciferase reaction where luminescence can be measured. The luminescence signal is proportional to the amount of ADP formed. Measurement is taken at reaction completion, after the reaction has been stopped.
While kinetic measurements with the ADP-GloTM Kinase Assay are less common, it is possible to choose to follow the reactions using kinetic mode or endpoint measurements in a microplate reader equipped with injectors. This is illustrated in the application note Promega’s ADP-Glo kinase assay.
Kinetic assays
While endpoint measurements provide a snapshot in time for analysis, researchers often want to repeat measurements over time to build up a picture of what is happening in a reaction or assay. Kinetic assays are used to measure the reaction rate over time, providing valuable information about enzyme activity and assay kinetics. In a microplate reader, two modes (plate mode and well mode) are possible which permit measurements broadly divided into the categories of slow or fast kinetic measurements, respectively. These kinetic assays are also known as continuous assays, as they allow real-time monitoring of the reaction progress until completion.
Kinetic methods can measure specific components in a complex sample with less interference compared to endpoint methods.
Plate mode
In plate mode for slower kinetic assays measurements are performed in cycles. In each cycle, each selected well of the microplate (as for endpoint measurements) is read once before the next cycle is started to repeat this process according to the number of defined cycles. These measurement cycles are typically taken at a specified time period to monitor changes in the reaction. For consistent results in repeated measurements, stable calibration standards or factors are often used, allowing for reliable calibration across multiple runs.
Bacterial and microbial growth assays
Plate mode is often used to measure bacterial growth and yeast growth on a microplate reader.
In the application note Determination of Minimum Inhibitory Concentration (MIC) of antibiotics using OD600 real time analysis in plate mode was used to determine the lowest concentration of an antibiotic that inhibits microbial growth, a useful parameter for many applications including studies of antimicrobial resistance (Fig. 3). High throughput capabilities and incubation options offer significant savings in time, efficiency and reliability for these types of measurements which can be made in plate mode.
Cell migration assays
Scratch (wound healing) migration assays also benefit from plate kinetic mode measurements. In these assays, cells are seeded into the wells of a microplate. A scratch is made across each well and cells migrate into the wound over time. The degree of wound closure can be monitored using fluorescence for stained or labelled cells or luminescence for metabolic reporters like Cell-Titer-GloTM. In the application note Real-Time Cell Motility Tracking Increases the Throughput of Scratch Wound Assays a CLARIOstar® with Atmospheric Control Unit (ACU) was used a scratch wound assay was used to monitor migration dynamics with well-scan detection. Luminescence assays were performed in kinetic plate mode.
Enzyme kinetic assays
Many enzyme assays can also be performed in plate mode to measure their kinetics. These assays are used to determine the reaction rate of the enzyme under various conditions, such as different pH, temperature, or substrate concentrations. Kinetic parameters like Km, kcat, and Vmax can be calculated as described in the application note Lysine deacetylase activity monitored by a fluorogenic assay using the CLARIOstar. Enzyme activities can be measured to allow for assay development suitable for measurements of the effectiveness of new drugs as described in the application note Assay development for essential enzyme activity in the tegument of live Schistosomes.
Pyrogen and endotoxin assays
The detection of pyrogens and endotoxins to ensure the safety of drug and medical device interventions can also be carried out effectively using plate mode measurements. In the application note Fast and highly sensitive pyrogen detection with the LumiMAT™ assay performed on BMG LABTECH microplate readers luminescence measurements were performed in plate mode for streamlined detection of endotoxins. You can read more about different pyrogen and endotoxin assays in our eBook Pyrogens: Past, present and future.
Oxygen radical absorbance capacity assays
Measurements of oxygen radical absorbance capacity or ORAC assays are also carried out in plate mode. One example is described in the application note ORAC assay to determine antioxidant capacity where the ability of antioxidants to neutralize reactive oxygen species (ROS) were investigated.
Well mode
In contrast to plate mode, well mode measurements are needed for reactions that take place with faster kinetics. In well mode, different measurements are made over time in the same well (up to 100 per second) and then the reader moves onto the next well for the next series of kinetic measurements. Spectrophotometry is often used in well mode to monitor reaction progress in real time by detecting changes in light associated with enzyme activity.
As mentioned earlier, some examples of where fast kinetic assays might need to be performed in well mode include investigations of the levels of biological signalling molecules like calcium ions, activity measurements of ion channels, studies of rapid enzyme kinetics, or measurements of essential biological processes like transcription. What all these processes have in common is that they occur on a fast time scale and require a series of reliable, accurate measurements to ensure the best results. Here are a few examples.
Ion and ion channel measurements
Calcium ions play a central role in many biological processes and are an important second messenger in cell signalling events. In many cases, researchers are interested in studying how the levels of calcium ions change in and outside the cell. Calcium flux assays detect intracellular calcium mobilisation in cells and follow the release of Ca2+ into the cytoplasm. When studying events such as synaptic transmission at the neuromuscular junction, an action potential arrives at the cell membrane, the membrane potential is depolarised, causing voltage-gated L-type calcium channels to open to allow an influx of Ca2+ into the cell. This leads to the release of Ca2+ from the sarcoplasmic reticulum, which also initiates a contraction by activating Ca2+-dependent contractile proteins.
In the application note Real-time calcium flux measurements in iPSC derived 3D heart tissue calcium flux was measured every 0.01 seconds using the dye Fluo-4 in well mode on the CLARIOstar®. It was possible to measure 100 data points per second in each well which was crucial to allow transient calcium measurements in real time to profile drug-induced changes relevant to the contraction of heart cells (in this case engineered heart cells were made from cardiac myocytes derived from human induced pluripotent stem cells) (Fig. 4).
Enzyme kinetic studies
Scientists often want to study how enzymes work on rapid timescales. We saw earlier that plate mode can be used to measure a range of different kinetic parameters for slow kinetic reactions. Well mode is more useful to determine kinetic parameters like Km, kcat, and Vmax when the speed of the reaction is much quicker. Rapid enzyme reactions may also benefit from the use of features like injectors which can deliver enzymes or reagents almost simultaneously with making the required detection measurement.
Transcription assays
Well mode is ideally suited to study the fast kinetic reactions that are used to study biological processes like transcription. The Dual-Luciferase® Reporter Assay or DLR for example is widely used to study gene transcription and regulation. The DLR assay is a two-step reaction that uses two luciferase enzymes that originate from Firefly and Renilla (Fig. 5). The Firefly reaction is first initiated and subsequently quenched before initiation of the Renilla reaction. Dual luciferase reactions are therefore fast reactions that require two injection steps. In these assays, researchers must make a series of measurements in the same well after the two injections have taken place. A microplate reader provides the ideal tool to make a series of these fast, repetitive measurements under different conditions.![Fig.5: Graph showing Firefly luciferase quenching taken from MARS evaluation software (>10,000 fold [n=24]. Data measured on an Omega series reader using 3.05 ng/ml of recombinant Firefly luciferase.](https://www.bmglabtech.com/hubfs/1_Webseite/5_Resources/Blogs/endpoint-and-kinetic-modes-fig5.webp)
Endpoint and kinetic mode assays on microplate readers
While we have looked at different assays that can be performed using endpoint and kinetic mode assays, it is also important to consider the features and technologies of a microplate reader that help to make these types of assays possible or which improve their performance.
Injectors
As we saw in the application note Dual-Luciferase® Reporter Assay or DLR, reagent injectors are often used to start or stop biochemical reactions. The ability to inject reagents and simultaneously detect a signal is particularly useful for reactions that have fast kinetics. Injectors are therefore great assets for reading in well mode since they offer the best performance in terms of speed and throughput (sampling rate). An injector should offer low dead volumes and the ability to back flush for precious reagents. BMG LABTECH microplate readers come with up to two injectors per reader and are compatible with plate formats with up to 384 wells.
Atmospheric Control Unit
Kinetic assays are often performed with cells that require specific growth conditions. Another useful feature for kinetic assays is therefore atmospheric control. The Atmospheric Control Unit from BMG LABTECH provides researchers with a system that uniquely enables control of both the oxygen and carbon dioxide concentrations in an independent manner.
Most microplate readers can control CO2 and O2 levels in the range of 1-20%. In some cases, they can decrease O2 to 0.1%. The CLARIOstar® Plus with ACU can reach 0.1% O2 and can read at 100 datapoints per second which makes it an ideal selection for plate and well mode measurements that require specific atmospheric conditions and rapid sampling. Atmospheric control is suitable for many applications including real-time cell-based assays, angiogenesis, cell viability, cell proliferation, cell migration, cell invasion, cytotoxicity studies and viral infection and neutralization studies.
Table 1. Overview of some BMG LABTECH application notes for endpoint and kinetic mode assays.
Endpoint assays
AN202: Promega's ADP-Glo kinase assay
AN269: Transcreener ADP2 FP assay certification for BMG LABTECH instrumentation
AN272: Overview of ELISA assays and NADH/NADPH conversion detection
AN299: Absorbance-based methods for protein quantification on BMG LABTECH instruments
AN363: Cytopathic effects of viruses for drug screening
AN369: Viable cell count detection assays
Kinetic assays
a) Plate mode
AN407: Determination of Minimum Inhibitory Concentration (MIC) of antibiotics using OD600
AN372: Real-Time Cell Motility Tracking Increases the Throughput of Scratch Wound Assays
AN292: Lysine deacetylase activity monitored by a fluorogenic assay using the CLARIOstar
AN315: Assay development for essential enzyme activity in the tegument of live Schistosomes
AN267: ORAC assay to determine antioxidant capacity
Well mode
AN253: Real-time calcium flux measurements in iPSC derived 3D heart tissue
AN301: GPCR second messengers in live cells
AN403: Peptide Ligands for Orphan GPCRs via Ca2+ Luminescence
AN396: Wastewater testing by A. fischeri luminescence
AN381: iPSC cardiomyocytes for calcium & metabolism assays
Future advances in endpoint and kinetic mode assays
The number of studies using endpoint and kinetic mode assays on BMG LABTECH microplate readers continues to grow at pace. New approaches and assays for endpoint and kinetic mode assays continue to be developed and each benefit from the selection of the most appropriate measurement be it a single data point when the reaction is over or continuous kinetic measurements during the reaction.
Whatever your requirements, BMG LABTECH has the microplate reader for your endpoint and kinetic mode assay applications.
The PHERAstar FSX was specifically conceived for screening campaigns and is your go-to reader for the fastest high-performance high-throughput screenings.
Both the VANTAstar® and CLARIOstar Plus allow for wavelength flexibility and include Enhanced Dynamic Range technology for superior performance in a single run of endpoint and kinetic mode assays. They also offer increased light transmission and sensitivity courtesy of Linear Variable Filter Monochromators™ and different filter options for endpoint and kinetic mode assays.
All BMG LABTECH microplate readers have exceptionally fast reading capabilities for endpoint and kinetic mode assays, which, together with the well mode function, ensure that sample reactions are recorded from start to finish without losing any important data points. In addition, the Omega series, VANTAstar, CLARIOstar Plus, and PHERAstar FSX microplate readers can be equipped with up to two reagent injectors, giving you full control over the addition of assay substrates and stimuli. During long-term kinetic experiments, microplate readers equipped with the ACU feature ensure that cell culture samples remain happy and that reliable and physiological measurement conditions are maintained throughout the entire measurement process.
Collectively, BMG LABTECH multi-mode readers combine high-quality measurements with miniaturised assays, short measurement times, and offer considerable savings on materials and other resources for endpoint and kinetic mode assays.
