Detection Application Notes

This technical note describes the successful performance of Promega´s Dual Luciferase Reporter Assay on the Tecan Infinite® M1000 multimode reader.
Material: Tecan Infinite® M1000 premium Quad4 Monochromators™ multimode microplate reader, equipped with a two channel injector and a luminescence module

• Dual-Luciferase® Reporter Assay System (Promega Corp., USA)
• QuantiLum® Recombinant Firefly Luciferase (Promega Corp., USA)
• Recombinant Renilla Luciferase (LUX Biotechnology, UK)

Tecan´s Infinite® M1000 premium Quad4 Monochromators™ multimode microplate reader has been validated and certified by Cisbio Bioassays, France for compatibility with the HTRF® technology. In this technical note we describe the implementation and instrument settings on the basis of validation experiments conducted with the nfinite® M1000 and the HTRF® Reader Control Kit, HTRF® cAMP assay and HTRF® cytokine (TNFα) assay from Cisbio.

This technical note describes how the Tecan HydroFlex™ platform was successfully evaluated for gentle washing of strongly adherent cell-line A431 as well as for the weakly adherent P815 cells in a 96-well format. For the cell-based assay described in this technical note, a HydroFlex platform, equipped with a standard wash head suitable for ELISA and cell washing, was used.

This technical note describes the successful performance of the Cambrex BioScience MycoAlertTM Assay on the Tecan InfiniteTM F500 filter based microplate reader. According to the achieved data, the performance of the instrument can be described as excellent.

This technical note describes the successful performance of Promega´s Dual Luciferase Reporter Assay on the Tecan Infinite® F500 multimode reader.

This technical note describes the successful implementation of HTRF® measurements on the Tecan Infinite® F500 filter based multifunctional reader. The Infinite F500 was proven to be a very well-suited detection platform regarding sensitivity and dynamic range of the investigated IL-1β detection - HTRF®assay kit.

The data obtained from the measurements reveal the correct separation of blue and green luminescent signals. The calculated values are comparable to those given by the kit manufacturer, PerkinElmer. They clearly underline the positive signal of the transfected and the negative signal of the non-transfected cells. Only slight variations between the use of a 96 well or 384 well microplates were observed, thus making the assay ready for higher throughput using the Tecan Infinite® F500 filter-based microplate reader.

This technical note describes the successful performance of Invitrogen´s PolarScreen™ Vitamin D Receptor Competitor Assay, Red on the Tecan Infinite® F500 filter based multimode detection system. In regard to the obtained data the instrument is perfectly performing according to the given assay requirements.

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.

This application note describes the successful implementation of the fluorescence intensity based Technothrombin® TGA assay on Tecan´s Infinite M200 monochromator based multimode detection system.
The amount of generated thrombin is dependent on the number of micro particles present in the sample. PFP showed a delayed and lower Thrombin formation compared to PPP.

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.

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.

The gain in the LS Scanner is a direct representation of the voltage of the PMT. It can be set to any value between 70 and 255. Because there is a linear relationship between gain and PMT voltage, this corresponds to PMT voltages between 330 and 1200 Volt.

We demonstrate here an example for the quantification of DNA, comparing absorbance (260 nm) with fluorescence detection using PicoGreenTM. We are able to show the advantage to have instrumentation available for both detection techniques, dependent on the yield of extracted DNA. The DNA samples used for the measurements were plasmid DNA and genomic DNA, extracted with the TECAN GENESIS RSP 150.

The present assay exemplifies a rapid and convenient way of monitoring H2O2 release associated with respiratory burst in freshly isolated or cultured leukocytes. The assay is microplatebased and utilizes the fluorescence generated from the administered HVA. The fluorescence signal can be detected with the SPECTRAFLUOR Plus microplate-fluorometer. The protocol is applicable to basic research as well as to clinically-related analyses of cells from diseased individuals.

The aim of all the various studies described in our application notes is to demonstrate the applicability of our microplate fluorometers (SPECTRAFLUOR or SPECTRAFLUOR Plus) for investigations using cell cultures including growth/ proliferation tests, cytotoxicity studies, studies on cell adhesion etc.

High-density plate washers can either be limited in flexibility, requiring dedicated systems specifically for cell washing or ELISA washing, or simply lack wash speed due to unproductive transport steps. To overcome these drawbacks, TECAN has developed the Power Washer 384 (PW384), an ultra-fast microplate washer combining cell washing and ELISA washing in 384-well and 96-well microplate formats.

LIVE/DEAD® Viability/Cytotoxicity Assay

Tecan Ultra Evolution, Safire and GENios Pro

Live cells are distinguished by the presence of ubiquitous intracellular esterase activity, determined by the enzymatic conversion of the virtually nonfluorescent cell-permeant Calcein AM to the intensely fluorescent Calcein. The polyanionic dye Calcein is well retained within live cells, producing an intense uniform green fluorescence in live cells (EX/EM ~495 nm/~515 nm). EthD-1 enters cells with damaged membranes and undergoes a 40-fold enhancement of fluorescence upon binding to nucleic acids, thereby producing a bright red fluorescence in dead cells (EX/EM ~495 nm/~635 nm). EthD-1 is excluded by the intact plasma membrane of live cells. The determination of cell viability depends on these physical and biochemical properties of cells. Cytotoxic events that do not affect these cell properties may not be accurately assessed using this method. Background fluorescence levels are inherently low with this assay technique, because the dyes are virtually nonfluorescent before interacting with cells (1). The LIVE/DEAD® Viability/Cytotoxicity Assay Kit (Molecular Probes, L-3224) provides a two-color fluorescence cell viability assay that is based on the simultaneous determination of live and dead cells with two probes that measure two recognized parameters of cell viability — intracellular esterase activity and plasma membrane integrity. Molecular Probes has found that Calcein AM and ethidium homodimer (EthD-) are optimal dyes for this application. The kit is suitable for use with fluorescence microscopes or fluorescence multiwell plate readers and easily adaptable for use with...

Detection of Calcein AM and Hoechst 33342

Tecan Ultra Evolution, Safire and GENios Pro

Calcein AM (acetoxymethyl ester of Calcein) is one of the premier indicators of cell viability due to its superior cell retention and the relative insensitivity of its fluorescence to physiological pH values. Live cells may be distinguished by the presence of ubiquitous intracellular esterase activity, determined by the enzymatic conversion of the virtually nonfluorescent cell-permeant Calcein AM to the intensely fluorescent Calcein. Calcein, which is the hydrolysis product of Calcein AM, is a polyanionic fluorescein derivative. Calcein is well retained within live cells, producing intense uniform green fluorescence in live cells (Molecular Probes: C-1430). Hoechst 33342, a bisbenzimide dye, is a cell membrane permeant, minor groove-binding DNA stain that...

Detection of Green Fluorescent Protein (eGFP)

Tecan Ultra Evolution, Safire and GENios Pro

spontaneously fluorescent protein isolated from coelenterates, such as the Pacific jellyfish, Aequorea victoria, or from the sea pansy, Renilla reniformis. Its role is to transduce the blue chemiluminescence of aequorin, into green fluorescent light by energy transfer. The molecular cloning of GFP cDNA and the expression of GFP as a functional transgene has opened exciting new avenues of investigation in cell, developmental and molecular biology. GFP can function as a protein tag, as it tolerates N- and C-terminal fusion to a broad variety of proteins many of which have been shown to retain native function. Highly specific intracellular localization including the nucleus, mitochondria, secretory pathway, plasma membrane and cytoskeleton can be achieved via fusion both to whole proteins and...

A New Generic Luminescent Protein Kinase Assay

Suitable For High Throughput Screening

Protein Kinases and their ability to phosphorylate proteins play key roles in the signal transduction pathways of many diseases such as cancer, arthritis and diabetes1. Thei importance of protein kinases makes them common targets for many High Throughput Screening departments (HTS) within the pharmaceutical industry. Current screening technologies such as HTRF and SPA employ the use of phospho-state specific antibodies or radioactive beads. The need for new antibodies and beads for each kinase/substrate pair makes these assays expensive and time consuming to develop and run. Cambrex have developed PKLight(™), a non-radioactive, homogeneous, robust and simple assay suitable for the screening of potentially all protein kinases in 96, 384 and 1536-well formats. This technology utilises Luciferase bioluminescence to measure ATP consumption as a result of kinase phosphorylation of the target substrate. The assay can be easily optimised for...

Fluorescence Lifetime (FLT)

A comparative report which demonstrates FLT efficacy in HTS

Relative to other detection/assay technologies

throughput screening (HTS), with the emphasis now shifted from numbers of compounds screened to the quality of the data and higher information content. This shift puts greater reliance on the assay or detection technology and its robustness in the screening environment.
Assay robustness is the term applied to how an assay signal tolerates interferences in a real screening campaign. Critical to this assessment are the performance parameters (Z’, signal window and reproducibility)1 by which assay quality of the control signals from an HTS are judged. But of equal importance, is the ability of the assay signal to avoid perturbation by the numerous non-specific effects that...

DNA Oligonucleotide Hybridisation

Automatic Detection of Fluorescence Lifetime with ULTRA Evolution

Hybridisation of two complementary DNA single-strands into one double strand is a fundamental biochemical reaction. The extent of this reaction can be inhibited or modulated by modifications of one of the binding partners (e.g. single nucleotide polymorphism) or by adding extraneous chemicals or proteins.
Within this technical note we utilise the well understood hybridisation reaction as a model system to demonstrate how to use Fluorescence Lifetime as a signal for monitoring (bio-)chemical reactions. Elsewhere we laid out the theoretical and technical basis for lifetime experiments (ref 1). In short, the light emitted from fluorescent labels is characterised by several parameters, among which one is the Fluorescence Lifetime. This parameter is highly susceptible to changes...

This application note deals with flash luminescence measurements performed on Tecan’s multifunctional injector plate reader GENios Pro by using a CHO mito-PhotinaTM based receptor-ligand assay.

The current technical note introduces two applications implemented and verified on the GENios Pro, one of Tecan’s multifunctional microplate readers. These applications utilise HTRF® (Homogeneous Time-Resolved Fluorescence) technique to generate the assay readout (CIS bio international, France).

The data above clearly show the ability of Tecan Ultra Evolution, Safire and GENios Pro to detect Calcein and EthD-1 and therefore to distinguish between live and dead cells with the bottom reading option of the instruments.We recommend the use of the multiple read per well (mrpw) function when performing LIVE/DEAD® measurements.

The data clearly show the ability of Tecan Ultra Evolution, Safire and GENios Pro to detect the two membrane permeant dyes, Calcein and Hoechst 33342, with the bottom reading option of the instruments.

Green fluorescent protein, GFP, is a spontaneously fluorescent protein isolated from coelenterates, such as the Pacific jellyfish, Aequorea victoria, or from the sea pansy, Renilla reniformis.The purpose of this technical note was:a) to determine the optimal filters for eGFP b) to compare the efficiency of a top and bottom measurement c) to compare Costar and Greiner plates d) to determine the ‘detection limit’ for transfection studies. All measurements were performed with Tecan Ultra Evolution, Safire and GENios Pro.

In this application note, we will describe the miRCURY LNA™ microRNA Arrays (Exiqon) and the HS Pro™ Hybridization Station (Tecan) and how they can be used for microRNA profiling of carcinomas of unknown primary origin. In addition, we will present some of the conclusions we have drawn from using the HS 4800 Pro™ Hybridization Station in conjunction with the miRCURY LNA™ microRNA Arrays.

Correct glycosylation is critical in the development of protein-based biopharmaceuticals. Procognia’s U-c Fingerprint products eliminate the need for sample preparation and provide quantitative data in approximately 5 hours enabling near real-time glycosylation monitoring. The combination of these assays with the Tecan HS 400 ProTM or the HS 4800 ProTM hybridization stations offers better possibilities for automation, which is a key factor for standardization, improving reproducibility and increasing throughput.

In this study manual methods with hybridization under coverslip were compared with the method using the HS 4800™ Hybridization Station concerning reproducibility, homogeneity and reducing of variability. For these purposes a typical manual procedure has to be translated into a protocol for the Tecan HS 4800.

In this study, we compared the assay performance of ISAC® allergen microarrays by employing two conceptually different methods: A manual assay operation and an automated assay procedure adapted for the Tecan HS 400.