TRF, TR-FRET & HTRF

Cytokine assays

Pro- and anti-inflammatory cytokines play a central role in autoimmune, inflammatory and infectious diseases. They are also key players in metabolic disorders and oncology, particularly the anti-tumor immune response. HTRF assays provide a versatile platform for the quantitation of various cytokines and chemokines and are suited to monitoring cytokine and chemokine release in cell-based experiments. With the SpectraMax i3x Multi-Mode Microplate Reader and SpectraMax® MiniMax™ 300 Imaging Cytometer, users are able to measure HTRF and cell viability, as well as confirm assay quality by monitoring cells’ appearance.

Explore our resources for cytokine assays:

• Application Note: Normalize HTRF cytokine assays to cell viability
• Application Note: HTRF Human TNFα Assay on SpectraMax Paradigm Multi-Mode Microplate Reader
• eBook: Explore cells from the inside out

eBook: From ELISA to epigenetics with HTRF

Overcome HTRF detection challenges with optimized microplate reader settings and simplified analysis.

• Easily enable a wide variety of HTRF assays
• Improve HTRF detection sensitivity with dedicated, validated microplate reader modules
• Get fast and simple analysis with dedicated HTRF protocols in SoftMax® Pro Software

eBook: From ELISA to epigenetics with HTRF

HTRF cAMP assays, Gαi/o-coupled receptors

Cyclic AMP (cAMP, cyclic adenosine 3’, 5’–monophosphate) is a key second messenger in GPCR signaling. The generation of cAMP is probably the most studied of signal transduction pathways and is involved in responses to sensory input, hormones, nerve transmission, and much more. Upon ligand binding to a GPCR, a conformational change occurs, activating the receptor and in turn activating a G protein. Activation of G_αi/o–coupled receptors inhibits adenyl cyclase from generating cAMP.

Explore our resources for G_αi/o

• Application Note: HTRF cAMP dynamic 2 and IP-One assays on the SpectraMax M5e reader
• Poster: Homogeneous High Throughput Live Cell GPCR Functional and Surface Binding Assays

HTRF cAMP assays, Gαs-coupled receptors

Cyclic AMP (cAMP, cyclic adenosine 3’, 5’–monophosphate) is a key second messenger in GPCR signaling. The generation of cAMP is probably the most studied of signal transduction pathways and is involved in responses to sensory input, hormones, nerve transmission, and much more. Upon ligand binding to a GPCR, a conformational change occurs, activating the receptor and in turn activating a G protein. Further signal transduction depends on the type of G protein activated, but activation of G_αs leads to upregulation of cAMP by adenylate cyclase.

Explore our resources for G_αs

• Application Note: HTRF cAMP HiRange Assay on SpectraMax Multi-Mode Microplate Readers
• Application Note: Detect GPCR activity with the cAMP-G_s HiRange HTRF assay
• Poster: Homogeneous High Throughput Live Cell GPCR Functional and Surface Binding Assays

HTRF assay

HTRF assays from Cisbio have evolved to encompass the areas of G protein-coupled receptors, kinases and cell signaling, epigenetics, and biomarkers. A wide variety of toolbox reagents and kits are available to address protein-protein interactions, nuclear receptor assays, receptor dimerization, ligand binding, receptor internalization, enzyme assays, and many more.

HTRF involves the use of two fluorophores, a donor and an acceptor, to label proteins or other biomolecules of interest. When the biomolecules interact, their proximity enables fluorescence resonance energy transfer (FRET) to occur from the donor to the acceptor fluorophore. Using long-lifetime fluorescent lanthanide cryptates as donors allows time-resolved detection of HTRF assays, minimizing short-lived background fluorescence from compounds and other materials. The detection of time-resolved FRET (TR-FRET) with a microplate reader provides a readout of biomolecular binding.

• Poster: HTRF technology on the SpectraMax i3 Multi-Mode Platform

HTRF-compatible microplate readers

Getting the best HTRF results requires a microplate reader that has been validated to meet the HTRF performance specifications set by Cisbio. A reader’s optics, energy source, and detectors, as well as the settings used to detect HTRF, all impact performance. Our readers use optimized components for HTRF detection, including an HTRF detection cartridge for the SpectraMax i3x and Paradigm Multi-Mode Microplate Readers, and an enhanced TRF module plus validated filters for the SpectraMax iD5 reader.

Cisbio’s HTRF certification program guarantees that readers bearing the “HTRF compatible” logo meet the specifications required for optimal HTRF performance. This technical note presents the optimized settings validated by Cisbio for Molecular Devices HTRF-compatible microplate readers.

• Application Note: Optimized HTRF settings for SpectraMax multi-mode microplate readers
• Application Note: HTRF profiling services on SpectraMax iD5 Multi-Mode Microplate Reader

IP-One assays, Gαq/11-coupled  receptors

Phospholipase C (PLC) is another common target of activated G proteins and is critical to a variety of processes. For example, thrombin receptors in platelets use this pathway to promote blood clotting. Cisbio IP-One competitive immunoassays detect the accumulation of inositol monophosphate (IP1), a stable downstream metabolite of IP3 induced by the stimulation of PLC following G_αq/11-coupled receptor activation. This kit allows direct characterization of receptor agonists and antagonists, in both adherent and suspension cells.

• Application Note: HTRF cAMP dynamic 2 and IP-One assays on the SpectraMax M5e Multi-Mode Microplate Reader

Kinase & epigenetic assays

Protein kinases are enzymes that play a central role in various signal transduction pathways involved in the control of cell growth, metabolism, differentiation, and apoptosis. Kinases are the second most important group of drug targets after GPCRs. Epigenetics is the study of heritable phenotype changes that do not involve alterations in nucleotide sequences, for example, changes such as DNA methylation and histone modification. These have been linked with the development of various diseases, particularly in cancer.

Explore our resources for kinase & epigenetic assays:

• Poster: High throughput homogeneous epigenetics assays using HTRF technology and the SpectraMax Paradigm microplate detection platform
• Poster: Homogeneous high throughput live cell GPCR functional and surface binding assays

Measure oxidative metabolism and glycolytic activity

To survive, cells require fuel in the form of ATP to carry out most essential bioprocesses. This fuel is generated through glycolysis and mitochondrial respiration. While both produce ATP, glycolysis can function in the absence of oxygen, whereas mitochondria require oxygen for the final step in oxidative phosphorylation (OXPHOS). Understanding how these pathways respond to effector compounds can provide useful insights into the overall function of cells and the underlying mechanisms that determine cell fate.

Read our application note to learn about real-time data acquisition and analysis of the progression of oxygen consumption and glycolic activity:

• Application Note: Measure oxidative metabolism and glycolytic activity on the SpectraMax i3x reader

Optimized settings for Transcreener TR-FRET assays

The Transcreener® TR-FRET Assays are single step, competitive immunoassays for direct detection of nucleotides with a far-red time resolved Förster resonance energy transfer (TR-FRET) readout. Learn how to optimize settings for Transcreener TR-FRET assays on the SpectraMax iD5 and i3x multi-mode microplate readers.

• Application Note: Optimized settings for Transcreener TR-FRET assays on SpectraMax iD5 and i3x readers