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The new Infinite readers offer affordable high performance detection solutions, powered by monochromator- and filter based technologies.
Access to a full range of leading detection methods with six application-tailored configurations Based on the acclaimed and proven Infinite 200 PRO reader, these new six upgradeable Infinite configurations provide a full range of leading detection methods in one easy-to-use modular instrument. Users can select from 6 configurations and options listed in the table below to create a perfect reader for their needs and budget.
Dynamic interactions between proteins are key mediators of multiple cellular functions and are involved in almost all biological processes. Accordingly, they are valuable targets for the development of novel drug therapies. In this context, bioluminescence resonance energy transfer (BRET), developed by Promega, is a well-established detection technology for the investigation of protein-protein interactions in living cells. However, its application has so far been somewhat hampered by its limited dynamic range and sensitivity.
One of the most used methods for determining nucleic acid concentrations is based on measuring the optical density of a sample at 260 nm (OD260). According to the Lambert-Beer law, the amount of absorbed light is proportional to the concentration of the sample, and to the pathlength the light has to pass through when going through the sample.
Implementation on Tecan’s Infinite® multimode reader series
Adenosine diphosphate (ADP) is an important mediator of cellular metabolism that is essential for maintaining energy levels. The Transcreener ADP2 TR-FRET Red Assay is intended for the detection of ADP production by any kinase or ATPase, and has been developed specifically for high throughput screening applications. The assay is based on the competitive binding of ADP to a monoclonal antibody-terbium conjugate, and uses a homogeneous TR-FRET detection mechanism (1). In the absence of free ADP, excitation of terbium results in energy transfer to a far-red tracer and emission at 665 nm. Any ADP produced by the enzyme of interest displaces the tracer from the antibody, disrupting the FRET reaction (see Figure 1).
BacTiter-Glo™ Microbial Cell Viability Assay measured on the Infinite® 200 PRO multimode microplate reader
The BacTiter-Glo Microbial Cell Viability Assay is a luminescence-based assay for the determination of the viability of microbial cells, using quantification of ATP as an indicator of metabolically active, viable cells (1). The chemistry relies on the properties of a thermostable luciferase (Ultra-Glo™ Recombinant Luciferase, Figure 1) and a proprietary formulation for extracting ATP from bacteria. The assay protocol comprises the addition of the BacTiter-Glo reagent directly to the sample, and subsequent measurement of luminescence; cell washing, removal of culture medium, and multiple pipetting steps are not required (Figure 2). The luminescent signal generated is proportional to the amount of ATP, and therefore an indicator of the number of viable cells in the sample. The ‘glow-type’ nature of the assay permits signal measurement over a period of approximately 30 minutes, depending on the type of bacteria and growth medium. The BacTiter-Glo Microbial Cell Viability Assay was tested on the Infinite F200 PRO filter-based multimode reader, using the instrument’s highly sensitive luminescence module.
We demonstrate the use of a non-activity-based cytotoxicity probe, CellTox™ Green Dye, which can be added at the beginning of an experiment and employed in real time
for the kinetic determination of cytotoxicity. We used the HP D300 Digital Dispenser, available from Tecan, for noncontact dispensing of test compound and the Tecan Infinite® M200 PRO with Gas Control Module (GCM™) for kinetic measurement of cytotoxicity in HepG2 cells over 72 hours.
In vitro cytotoxicity is largely influenced by test article concentration and the exposure period with cells. The diversity of kinetic response can complicate conventional cytotoxicity endpoint assay determinations because most assay reagents are formulated to measure enzymatic biomarkers that are susceptible to time-dependent decay. Using DNA binding as an indicator of cytotoxicity mitigates this issue, providing a robust method of detection for time course experiments. CellTox™ Green Dye is an asymmetric cyanine
dye that measures cytotoxicity as a result of compromised cellular membrane integrity (Figure 1). When the dye binds to DNA, green fluorescence is enhanced. The signal intensity is directly correlated to the degree of change in the membrane integrity. The dye can be added at the time of cell plating or compound treatment and the fluorescence monitored kinetically over a period of up to 72 hours. Alternatively, the dye can be added at the conclusion of compound treatment and an endpoint measurement taken.
Measuring oxygen radical absorbance capacity with the Infinite® 200 PRO multimode reader
Reactive oxygen species (ROS) are generated as natural byproducts of the cellular metabolism. They are involved in various biological processes, functioning as important signal mediators. However, excess intracellular levels of ROS may result in cell and tissue damage, and are associated with degenerative diseases, most notably cancer. In healthy individuals, intracellular antioxidant systems maintain ROS levels below a critical threshold, permitting essential ROSmediated signaling processes to function, but preventing ROS overproduction and potential tissue damage . Cells that fail to compensate and neutralize heightened ROS levels die by apoptosis to avoid passing on ROS-caused DNA damage to daughter cells. Any dysfunctions in the cellular antioxidant systems can therefore have serious consequences. In addition to the cells’ own antioxidant systems, various studies have suggested a relationship between an antioxidant-rich diet and a good health status, implicating that the consumption of antioxidant-containing foods can help to maintain health and even prevent certain diseases .
A well-established and reliable method to determine the antioxidant capacity of a substance is the oxygen radical absorbance capacity (ORAC) assay . It is based on the inhibition of oxyradical-induced oxidation of 2,2’-azobis-(2- methylpropionamidine) dihydrochloride (AAPH) by substances with antioxidant properties. Peroxyl radicals produced in a time-dependent manner during the thermal decomposition of AAPH will quench the fluorescence signal. In the presence of a substance with antioxidant properties the fluorescence reduction is inhibited, depending on the substance’s ORAC capacity. The dynamics of the signal inhibition, expressed as the area under the curve (AUC), are used to quantify the antioxidant capacity, expressed as the ORAC value, by comparing the sample AUC to an antioxidant standard curve generated with Trolox, a water-soluble vitamin E analog. This application note describes the use of the Infinite® F200 PRO in combination with a commercially available ORAC assay kit, using different beverages as antioxidant samples...
Fluorescence-based drug sensitivity testing using 3D tumor microtissues and the Infinite® M200 PRO monochromator-based multimode reader
Cell viability assays are commonly used in cell biology and drug discovery to characterize cell responses to endogenous and exogenous factors or substances, such as cytotoxic drugs and environmental changes (1). Generally, cell viability is assessed using vital dyes, from which viability can be concluded either directly or indirectly. This method, although often used, is labor intensive and tedious. Automated alternatives include electric cell counters (1) and flow cytometers which, although accurate, are associated with sophisticated equipment and high assay costs, and require technical expertise. As a result, fluorescence-based cellular assays are becoming increasingly popular, due to their sensitivity and versatility.
The Infinite M200 PRO offers enhanced fluorescence intensity reading for cell-based and biochemical applications, with a range of features designed to improve sensitivity and inter- /intra-well reproducibility. Functions such as orbital shaking, temperature control and enhanced fluorescence bottom reading with the optimal reading (OR) function ensure excellent performance and reliability.
To further improve the predictive power of in vitro cell-based assays, cell models have to mimic more closely the three dimensional (3D) structure of organs and tissues in vivo (2). Scaffold-based 3D cell culture approaches often suffer high background fluorescence, due to autofluorescence from the scaffold biomaterials. Scaffold-free microtissues are therefore ideally suited to this application, offering tissue-like structures while allowing researchers to take advantage of embedded fluorescent reporter technology.
Combining Tecan’s Infinite M200 PRO with InSphero’s organotypic microtissue tumor model (which harbors fluorescent reporter proteins) provides a scalable system to assess drug sensitivity in complex 3D cell culture models. This allows long-term, tissue-based analyses – such as cell proliferation studies – offering reproducible measurements over time....
Walkaway growth monitoring of Helicobacter pylori in the Infinite® 200 PRO reader with Gas Control Module (GCM™)
Growth analysis of microorganisms via OD absorbance measurements at 600 nm is key to many different research areas. In the past, this has been a time-consuming and
labor-intensive procedure. Tecan’s Infinite 200 PRO multimode reader can now provide all essential growth conditions – including continuous shaking, temperature control, O2 and CO2 regulation and humidity stabilization – within the measurement chamber, eliminating the need to transfer microplates between the cell incubator and the microplate reader (1). This enables simultaneous incubation and signal detection without the need for any manual intervention, even for microorganisms which need very specific environmental conditions, such as facultative anaerobic bacteria.
In this collaborative study, Tecan and its partners have analyzed the growth of the human pathogen and class-I carcinogen Helicobacter pylori over a period of 28 hours.
As a microaerophilic organism, H. pylori needs low atmospheric oxygen concentrations for optimized growth, and normally colonizes human mucosa, where it can cause certain
types of stomach disorders and cancer. To mimic these physiological conditions, it is a prerequisite for the incubation/detection device to have the capability to control atmospheric O2 levels.
Bacteria were incubated at 37 °C inside the measurement chamber of the Infinite 200 PRO reader, with continuous shaking, 10 % CO2 and varying O2 levels (normoxic (control), 5 % and 10 % O2). The proliferation was monitored by measuring sample absorbance at 600 nm, and the fluorescence of GFP-transformed H. pylori. To minimize
evaporation effects during the long incubation period inside the reader, Tecan recommends using the Nunc Edge 96-well plate, which has been shown to minimize evaporation when used in combination the Infinite 200 PRO and the GCM (2, 3)...
Detection of tyrosine kinase activity using the Infinite F200 PRO’s new AlphaScreen function
AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay) is a bead-based screening technology developed for fast, reliable and cost-effective detection of biomolecular interactions. It utilizes the energy transfer between donor and
acceptor beads that occurs when these are brought into close proximity due to a binding event between their coupling partners. As a result, a strong luminescent signal is generated that can be detected in a wavelength range of 520-620 nm .
Tyrosine kinases are important mediators of cellular processes such as signal transduction, cell growth and apoptosis. They have been reported to be involved in a
number of diseases associated with excessive cell proliferation – including atherosclerosis and cancer – and are therefore often targeted in drug development and highthroughput screening (HTS) approaches.
AlphaScreen-based phosphotyrosine assay kits, for example P-Tyr-100, have been developed for reliable and sensitive detection of kinase activity. The AlphaScreen signal is dependent on the extent of tyrosine kinase phosphorylation..
The Infinite F200 PRO is one of Tecan’s most reliable and sensitive filter-based multimode readers. It features all common measurement modes, including absorbance, fluorescence top/bottom, single and dual luminescence, fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET) techniques such as HTRF®. In addition, the Infinite F200 PRO is now capable of measuring AlphaScreen- and AlphaLISA®-based assays...
Detection of human immunoglobulin G (IgG) using the Infinite F200 PRO’s new AlphaLISA function
AlphaLISA is a homogeneous, no-wash alternative to conventional ELISAs based on PerkinElmer’s bead-based Alpha (Amplified Luminescent Proximity Homogeneous
Assay) technology. AlphaLISA assays can be set up as sandwich or competitive immunoassays to detect and quantify molecules of interest in biological samples .
High energy excitation of photosensitizer molecules within the AlphaLISA donor beads at 680 nm converts ambient oxygen to singlet oxygen, which in turn is able react with the chemistry in the acceptor beads if these are in close proximity. A cascade of energy transfer steps ultimately results in the generation of a strong luminescence signal at 615 nm, indicating specific binding between the molecules attached to the two bead types. The fluorophores embedded in the AlphaLISA acceptor beads produce a narrower bandwidth signal than the acceptor beads used for classical AlphaScreen® assays.
This makes AlphaLISA assays less prone to signal interference at wavelengths of <600 nm, increasing the sensitivity and robustness of the assay. The use of dedicated AlphaLISA optics permits the analysis of target molecules in blood and serum by drastically reducing the effect of hemoglobin within a sample.
The new module for AlphaScreen and AlphaLISA assays is the latest addition to the Infinite F200 PRO’s multimode functionality. In addition to established reading modes,
including absorbance, fluorescence top/bottom, single and dual luminescence, fluorescence polarization (FP) and timeresolved fluorescence resonance energy transfer (TR-FRET) techniques such as HTRF® and LanthaScreen™, the Infinite F200 PRO now offers a module for Alpha-based assays, tailored to the needs of low to medium throughput applications and basic research...
Fluorescence-based drug sensitivity testing using 3D tumor microtissues and the Infinite® M200 PRO monochromator-based multimode reader
Optimizing bacterial growth studies on the Infinite® 200 PRO multimode reader platform using permanent shaking and heating
Long-term cell-based kinetics using Tecan’s GCM™ and the Thermo Scientific Nunc Edge plate
Enabling long-term cell-based assays with eukaryotic cells via controlled CO2 partial pressure inside the reader
Cell-based applications are central to life science research. They may range from cytotoxicity, proliferation, apoptosis and GPCR signaling assays to high-throughput screening (HTS) drug discovery applications. Adherent cell types are typically analyzed through the well bottom in order to bring the cell monolayer as close as possible to the detector and avoid unspecific fluorescence noise by the overlying growth medium. For this reason it is essential to guarantee illumination and reading of the entire well bottom, since cells are not always homogeneously distributed over the growth surface.
In vivo kinetic studies of drug uptake across the gastro-intestinal tract (GIT) and blood-brain barrier (BBB) are valuable tools for assessing bioavailability to prospective targets. These are relatively expensive and time consuming assays which are conducted sparingly. pION Inc. has introduced a parallel artificial membrane permeability assay (PAMPA) which has recently gained popularity as a novel, cost-effective high-throughput assay capable of rapidly screening compounds for their permeability characteristics in early drug discovery. In this note we describe the easy implementation of Tecan´s Infinite M200 with its absorbance scanning feature for PAMPA sample analysis.
Here we describe the preparation and evaluation of RNA samples from a novel population of mouse keratinocyte stem cells marked by Lgr5+ expression. We used tools specifically designed for handling and measuring low RNA quantities including RNA purification and the subsequent quantification with the Infinite® M200 NanoQuant.
In this application note we describe the use of Tecan’s HydroFlexTM washer equipped with a magnetic bead plate carrier for fast purification of a large number of samples using magnetic beads.For the detection of the luminescence signal Tecan’s Infinite® F200 multimode reader was used.
Among the devices suitable for measurements of small-volume samples Tecan’s Infinite® 200 NanoQuant in combination with the NanoQuant Plate™ offers uncompromised performance for absorbance-based nucleic acid quantification and assessment of labeling efficiency and, in addition, can be upgraded at individual convenience with fluorescence and luminescence reading functions.The present note describes the implementation of the NanoQuant Plate™ for protein measurements with regard to essential assay parameters such as linearity, uniformity, and reproducibility.
Protein quantification is often required before proceeding with protein samples for isolation, chromatographic or electro-phoretic analysis, or immunohistochemical methods. Two different techniques are generally used for colorimetric detection and quantification of proteins: protein-dye binding and protein-copper chelation. In this note we describe the use of Tecan’s Infinite® F200 and Infinite M200 instruments for easy and sensitive protein quantification using different absorbance-based assays.
For protein assays in general, and especially for the modified Lowry protein assay, the reader-injector system is an ideal solution avoiding pipetting and workflow errors, and resulting in consistent and traceable data.Tecan´s Infinite® 200 instrument series extended with the injector system offers a multi-functional system, from injection of reagents, to mixing, incubation and measuring, with all steps performed within one instrument set-up.
Implementation on Tecan’s Infinite® M200 PRO multimode reader
In recent years genetic reporter systems have greatly influenced analysis and understanding of gene expression, gene regulation and cellular responses in both eukaryotic and prokaryotic cells. A genetic reporter system consists of a promoter or a genetic element under analysis joined to a reporter gene in an expression vector. Expression of the reporter protein can be accomplished by measuring the protein itself or the enzymatic activity of the protein.
Comparing luminescence measurements using the Photon-Counting Tube-based Infinite® M200 PRO with Photomultiplier Tube-based multimode readers
Multimode microplate readers (MMRs) are commonly equipped with photomultiplier tubes (PMTs). PMTs are extremely sensitive detectors of light in the ultraviolet, visible and near-infrared ranges of the electromagnetic spectrum. A PMT multiplies the photocurrent produced by incident light by as much as 100 million times, enabling individual photons to be detected (1). PMTs in multimode readers are mainly used for fluorescence- and luminescence-based measurements, and can be run in two modes; photomultiplier tube mode (PMT mode) and photon-counting tube mode (PCT mode). This mode cannot be defined by the user, and is set at the manufacturing site.
In PMT mode, which is commonly used for fluorescence measurements, the amplification rate of photocurrents – the ‘gain’ of the PMT – can be controlled by the user through the instrument’s software. A low gain value (low amplification) is used to measure high assay signals, whereas a high gain value (high amplification) is used to detect low assay signals.
Virtually all MMRs on the market are also equipped with a feature to automatically determine the optimal gain level for an assay plate based on the highest signal on the plate. As the final data values are always related to the gain level applied, they are given as relative units (RFU, relative fluorescence units).
For smaller photon fluxes, such as luminescence applications, the photomultiplier can be operated in PCT mode, where final data is given as absolute values in counts per second (cps). In this mode, the photomultiplier gain is set to a fixed level by the manufacturer so that a single photoelectron (resulting from a single photon incident on the primary surface) generates a very large current at the output circuit. This predefined gain setting guarantees maximum sensitivity, but the user cannot control the amplification rate, thus not preventing the detection of high signals which are outside the range of the PMT (‘OVER’ signals).
Comparing absorbance measurements between the Quad4 Monochromators™-based Infinite® M200 PRO and a multimode reader using photodiode array technology
The most widely established technology for UV-Vis absorbance measurement is a monochromator-based microplate reader. Spectrophotometers have undergone a great deal of development since their introduction in the early 1950s (1) and, in recent years, multimode readers using linear photodiode array (PDA) technology for absorbance measurements have become available. PDA-based readers incorporate an optical grating and a solid state array detector, enabling measurement of light intensity throughout the UV and visible regions of the spectrum. Similar to a monochromator, but much faster, they allow the entire UV-Vis spectrum of a sample to be captured within a few seconds per well. However, this technology suffers from a number of drawbacks, mainly due to high levels of stray light. This results in a dramatically limited dynamic measurement range (2). This technical note compares the results of basic absorbance measurements performed on an Infinite M200 PRO multimode reader equipped with Quad4 Monochromators with those from a multimode reader equipped with a PDA.
Sensitivity uniformity of AlphaScreen measurements
AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay) is a bead-based screening technology developed for fast, reliable and cost-effective detection of biological interactions. The AlphaScreen chemistry employs donor and acceptor beads that can be attached to various types of biologically relevant molecules. The phthalocyanine photosensitizer molecules embedded in the AlphaScreen
donor beads convert ambient oxygen into singlet oxygen when excited at 680 nm. The singlet oxygen molecules are able to cover a distance of up to 200 nm during their half-life of approximately 4 μs. If AlphaScreen acceptor beads are in close proximity to the donor beads, due to biological binding of their coupling partners, the singlet oxygen molecules are able to initiate a cascade of energy transfer steps in the acceptor beads, ultimately resulting in the generation of a strong light emission in the range of 520-620 nm. Due to the amplified signal, even small amounts of biological analytes can be detected .
The Infinite F200 PRO is one of Tecan’s most reliable and sensitive filter-based multimode readers. It features all common measurement modes, including absorbance, fluorescence top/bottom, single and dual luminescence, fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET, including HTRF®). In addition, the Infinite F200 PRO is now capable of measuring AlphaScreen- and AlphaLISA-based assays.
The Infinite F200 PRO uses its fluorescence module in combination with a dedicated dichroic mirror and optimized filter sets for AlphaScreen and AlphaLISA applications. While the excitation is performed at 680 nm in both cases, the emission filters are different for AlphaScreen and AlphaLISA readings; AlphaScreen signals are recorded using a 570 (100) nm filter, and AlphaLISA measurements use a 615 (20) nm
filter that minimizes hemoglobin-caused assay background...
Determination of instrument detection limits using the Glowell™ GLO-466 standard
Enhanced Assay Performance using the Tecan Infinite® M200 Multimode Reader
Enhanced Assay Performance using the Tecan Infinite® M200 PRO Multimode Reader
Enhanced performance using the Tecan Infinite® M200 multimode reader
Enhanced performance using the Tecan Infinite® M200 multimode reader
Sensitive detection using the Tecan Infinite® M200 multimode reader
Enhanced performance using the Tecan Infinite® M200 multimode reader
Technical details and descriptions of applied features
Optimizing the luminescence sensitivity in the Infinite 200 PRO multimode reader series
Optimizing TRF sensitivity in the Infinite 200 PRO series multimode readers
Optimizing the FI bottom sensitivity in the Infinite 200 PRO multimode reader series
Optimizing the FI top sensitivity in the Infinite 200 PRO multimode reader series
Based on the successful Infinite 200 series, Tecan has developed the Infinite 200 PRO, with new enhancements, like the automated adjustable z-focusing in the Infinite M200 PRO. The adjustable z-focus for FI top measurements, which offers equally high sensitivity for all plate formats, helping to implement assay miniaturization. It is a flexible tool to customize measurement parameters to plate type and assay volume Z-focusing can be performed either by using the automated zcalculation function in the fluorescence intensity measurement stripe or by selecting the z-position option of the instrument menu in i-controlTM software. The z-position dialog of the instrument menu of Infinite M200 PRO provides an additional function which allows performing z-position scans for both a signal and a blank well for each fluorescent top measurement label. Beside the optimal z-position of signal and blank, the zposition for the maximum signal to blank ratio is also displayed. This automated z-adjustment with integrated background value correction is particularly suited to cell-based applications using autofluorescent growth media, providing automatic optimization of the signal-to-background ratio. The desired z-positions can be selected and applied to the measurement script by the user. In this technical note the impact of z-focusing on the sensitivity of fluorescence top measurements on Infinite M200 PRO is presented.
Implementation on Tecan’s Infinite® 200 Multimode Reader Series
This Technical Note describes the successful<br>
implementation of Invitrogen’s LanthaScreen TR-FRET assay system on Tecan’s Infinite F200 filter-based multi modular detection system. The Infinite F200 has performed according to Invitrogen’s LanthaScreen certification program criteria and was successfully validated by Invitrogen as “LanthaScreen® Certified”.
Cell proliferation and viability assays are commonly used to assess cell number and cytotoxic effects. In this study the performance of the Infinite M200 with respect to the MTT assay, CellTiter 96 AQueous One Solution Cell Proliferation Assay and CellTiter-Blue Cell Viability Assay, respectively, was evaluated.
Scientists from Affymetrix® Inc., a world-leading supplier of microarray equipment and assays, evaluated the Tecan Infinite® 200 NanoQuant multimode microplate reader and two other spectrophotometers for DNA concentration analysis.
In this note, data are presented from a comparison done by Affymetrix using Tecan’s Infinite 200 NanoQuant, Molecular Devices SpectraMax® Plus and the Nanodrop® ND1000 instrument.
For measurement and differentiation of small amounts of RNA and DNA, two detection systems using fluorescent nucleic acid binding dyes have been tested - the Quant-iT PicoGreen® reagent to measure DNA and the RiboGreen® reagent for detection of small amounts of RNA.For detection and quantification of small amounts of dsDNA and RNA within a wide range of concentrations, the fluorescence intensity measurement with the Tecan Infinite® 200 series provides sensitive and accurate measurement results.
Cell-based assays are powerful and versatile tools for experimental design in life science research, and are frequently used for high throughput screening analysis. Cell
adhesion, chemotaxis, cell migration and invasion are essential topics in cancer development and tumor growth. Cell-based assays on micro-porous membranes provide
outstanding in vitro models for drug discovery in research areas such as angiogenesis, neoplasia and inflammation.
Principle of FluoroBlok cell culture inserts
BD Falcon FluoroBlok cell culture inserts allow easy and non-destructive analysis of invasion and migration assays, as well as offering automation compatibility (1). Fluorescently labeled cells inserted into the top chamber migrate through the membrane and are detected by bottom reading fluorescence plate readers and microscopes.
Cells present in the top chamber of the insert are shielded by a light-tight polyethylene terephthalate (PET) membrane that blocks light within the 490-700 nm range. BD Falcon
FluoroBlok cell culture inserts are available in different formats, pore sizes and protein coatings for various applications (1).
Inducing cell migration
In the initial experiments, migration of cells through the FluoroBlok filter was validated by inverted microscopy; however, cell quantification is constrained by its variability, due to cell spreading in the presence of serum, and by the fact that it is very time-consuming. To improve the throughput of this assay, quantification of cell migration was performed by measuring fluorescence intensities in the FluoroBlok bottom chambers using the Infinite M200 PRO multimode microplate reader...