Case Studies – ION Biosciences

Multiplexing GPCR Signaling Pathways with Spectrally Resolved Fluorescent Dyes

Miguel Torres — Fri, 05 Jun 2026 03:52:25 +0000

Case Studies

Simultaneously measure calcium signaling and GIRK activation using Fluo-Gold and Thallos on the CLARIOstar® Plus.

Understanding the complex interactions of G protein-coupled receptor (GPCR) signaling pathways is critical for advancing drug discovery and biomedical research.

In this collaborative application note, ION Biosciences and BMG LABTECH demonstrate the successful detection of two distinct GPCR signaling casecades using spectrally resolved fluorescent dyes. By combining Fluo-Gold AM and Thallos AM on the CLARIOstar® Plus platform, researchers can monitor calcium release induced by Gq agonism and potassium channel activitation by the beta/gamma subunits of Gi GPCRs in real time within the same well.

A multiplexed approach can help you uncover biased ligands and simultaneously promotes faster outcomes in your GPCR discovery campaign.

KEY BENEFITS

Monitor Multiple Signaling Pathways Simultaneously

Measure two distinct GPCR signaling pathways in the same well for more complete SAR.

Spectral Resolution

The PHERAstar FSX provides ultimate flexibility for discriminating between dyes.

Double the Data Output

Capturing two data points simultaneously saves time and resources.

Reduce Profiling Time

Generate richer datasets in fewer experiments to accelerate compound characterization and screening workflows.

Assay Principle

Cells expressing M3 and D2 receptors along with GIRK channels are loaded with Fluo-Gold AM and Thallos AM dyes.

Stimulation with acetylcholine activates the M3 receptor and triggers calcium release, while dopamine activates the D2 receptor and induces potassium flux through GIRK channels. Using spectrally resolved fluorescent dyes, both signaling pathways can be measured simultaneously in real time.

This approach can be translated to other GPCR secondary messenger and fluorescent sensor pairs within our portfolio.

Advance Your GPCR Research with Multiplex Signaling Analysis

Download the full application note to explore the experimental workflow, optimization strategy, and complete results.

Download Application Note (PDF)

Innovative Dyes for Ion Channel Research

High-quality fluorescent dyes designed for accurate and reliable measurements.

High Performance and Reliability

Trusted by researchers worldwide for consistent, reproducible results.

Trusted by Researchers Worldwide

Supporting scientific discovery in academia and industry globally.

Expert Support for Drug Discovery

Out team is here to help you succeed at every stage of your research.

Brilliant Thallium Snapshot kit validated for endpoint potassium assays

Miguel Torres — Thu, 28 May 2026 15:18:43 +0000

Case Studies

Discover how BMG LABTECH validated the Brilliant Thallium Snapshot kit for endpoint potassium channel screening on their PHERAstar FSX multi-mode plate reader.

Download Application Note (PDF)

In this collaborative Application Note with BMG LABTECH, ION’s Brilliant Thallium Snapshot kit demonstrated results comparable to kinetic imaging readers using the PHERAstar FSX multi-mode plate reader. This simplified endpoint assay workflow provides a cost-effective alternative for potassium channel screening without requiring specialized kinetic imaging systems.

Endpoint assay, simplified

Thallium flux measured in an endpoint format—no kinetic imaging reader required.

Comparable to FLIPR

Concentration-response results on the PHERAstar FSX are comparable to kinetic imaging (FLIPR) systems.

Optimized for HTS

Validated in 1536-well plates for high-throughput screening campaigns.

Cost-effective alternative

A more accessible solution that expands potassium channel screening to more labs.

Comparable performance to kinetic imaging readers

Read the full Application Note

Get the full experimental details, optimization data, and screening results in this collaborative study with BMG LABTECH.

Download Application Note (PDF)

Questions about this application?

Get the full experimental details, optimization data, and screening results in this collaborative study with BMG LABTECH.

Enquire Now

Comprehensive Assay Collection for TMEM175 Drug Discovery

Miguel Torres — Wed, 03 Dec 2025 19:40:38 +0000

Case Studies

TMEM175 as a Target for Drug Discovery

TMEM175 is a lysosomal ion channel that functions as both a proton channel and potassium channel. It plays a crucial role in maintaining lysosomal pH, and is essential for lysosomal function. Mutations in TMEM175 lead to impaired lysosomal degradation and increased neuronal susceptibility to alpha-synuclein aggregation, and are associated with an increased risk of developing Parkinson’s disease.

Validated TMEM175 Assays

Gain exclusive insights for your Parkinson’s disease pipeline with our suite of proprietary assays to identify TMEM175 inhibitors and activators. Our TMEM175 services are supported by the most expansive collection of HTS assays to deliver valuable information about how your compounds interact with this disease relevant lysosomal target. All assays generate EC50 values in line with literature reported values for known tool compounds, ensuring data reliability from day to day. Our validated cell line and robust assay platforms can be leveraged to assess what you need, from ion permeability profiling to inhibitor identification.

If you’re searching for TMEM175 variants or additional readouts to support your research, our team can help.

Thallium-free Potassium Channel Assay

Proprietary ION technology that measures potassium efflux through activated TMEM175 channels expressed on the plasma membrane in real time. DCPIB and 4-AP are used to demonstrate a robust hit identification capability. The measurement of potassium yields physiologically relevant data for TMEM175 when compared to other surrogate readouts.

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Brilliant Thallium Assay

Gold standard for potassium channel drug discovery. Thallium acts as a surrogate for potassium ions to probe TMEM175 channel activity. This solution is able to identify activators and inhibitors of TMEM175, but requires longer compound incubation times to generate a robust response.

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Brilliant Thallium Snapshot Assay

Proprietary ION technology that measures thallium efflux in response to TMEM175 activation. This assay produces the largest screening window of all available assays, and can be used to identify activators or inhibitors. TMEM175 activation is visualized in real time upon stimulation with DCPIB.

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Brilliant Intracellular pH Assay

Measures proton transport through TMEM175 upon activation with DCPIB. As a predominantly proton channel on its native lysosomal membrane, this physiologically relevant readout can help you identify compounds that alter the proton conductance of TMEM175.

Lysosomal TMEM175 Assays

Powered by our Halotag-compatible thallium indicator collection, we’ve enabled HTS of TMEM175 activity expressed on lysosomal membranes inside viable, intact cells. Available in 384-well plate formats for high-throughput drug discovery campaigns. No lysosomal manipulation or isolation required. Probe TMEM175 in its native membrane and environment to get the most relevant insights into target engagement and TMEM175 pharmacology.

Lysosomal Thallium Flux Assay

Our proprietary thallium dye is localized to the lumen of lysosomes to measure thallium flux across the lysosomal membrane in intact cells. Enables the identification of activators, such as DCPIB, and inhibitors, like 4-AP, of lysosomal TMEM175 using standard HTS methods (384-well format).

ION Thallos Gold-HTL loaded lysosomes

Sample activator and inhibitor data using our lysosomal drug discovery platform for TMEM175 assays. Included data are normalized kinetic traces (top left), a dose response curve to DCPIB (top right), and a dose response curve to 4-AP in TMEM175 expressing cells and wild-type cells (bottom left).

Lysosomal pH Assay

Immunofluorescence series of cells stained with pH-HTL, a TMEM175 antibody, and hoechst (a nuclear stain).

Using a custom pH-sensitive dye that is also a ligand for Halotag (pH-HTL), we can localize the pH indicator to the lumen of lysosomes. From there, we measure proton flux across the lysosomal membrane in intact cells. This technology enables the identification of activators, such as DCPIB, and inhibitors, like 4-AP, of lysosomal TMEM175 proton conductance using standard HTS methods (384-well format). The assay can be conducted using HTS instrumentation or high content imaging platforms for spatial resolution and realtime visualization of lysosomal pH.

Affect of Bafilomycin A on luminal lysosomal pH with TMEM175 overexpression and DCPIB treatment.

Timelapse of pH-HTL and TMR (pH-insensitive) labeled lysosomes after DCPIB treatment displaying an obvious shift from red to green, indicative of alkalization of the lysosomal lumen.

This platform can also be used to explore additional modulators of lysosomal pH, a crucial regulatory mechanism for lysosomal function, autophagy, and nutrient transport. Transporters including proton pumps (e.g. V-ATPase, SLC7A11), SLC transporters (e.g. SLC15A3 and SLC15A4), and lysosomal ion channels such as TPC2 (two-pore channel 2), all play a role in maintaining or using the lysosomal pH gradient.

Summary

Whether you are screening for TMEM175 modulators or exploring unique pharmacology, our TMEM175 platform offers proprietary insights, maximum throughput, readout flexibility, and physiologically relevant data to support your discovery needs.

Dive deep into TMEM175 pharmacology with us

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High-throughput technologies for Gi GPCR drug discovery

Manuel Pacheco — Tue, 02 Dec 2025 16:37:50 +0000

New Case Study:

Case Studies

Introduction

G protein–gated inwardly rectifying potassium (GIRK) channels strongly shape neuronal and cardiac excitability and, in the brain, mainly form Kir3.1/Kir3.2 heterotetramers activated by Gβγ released from Gi/o-coupled GPCRs, making GIRK activity a direct, physiological readout of Gi/o signaling. Traditional Gi/o assays such as FRET, BRET, or cAMP measurements are limited by pathway complexity or depend on artificial adenylate cyclase stimulation. Here we present a scalable Gi/o GPCR platform for high-throughput, real-time GIRK quantification using both fluorescence and automated patch clamp. The assay uses HEK293 cells stably expressing Kir3.1/3.2 and allows just-in-time transient expression of diverse Gi/o-coupled GPCRs. GIRK activation by ML297 produced comparable EC50 values across fluorescence and electrophysiology, and receptor-driven responses were absent in control HEK cells lacking M2 or GIRK. Overall, this method enables reproducible pharmacology across many neurotransmitter receptors and supports discovery of modulators of Gi/o-linked GPCRs.

Gi GPCR Assay Platform

Stable cell line over-expressing GiRK channels that enables:

Fluorescence and automated electrophysiology-based readouts bridge the gap from hit identification to validation workflows, all at high throughput
Transient GPCR expression using plasmids, lentivirus, or bacmam vectors for maximum flexibility and fast turnaround times

This cross-platform screening technology gives reliable results to accelerate your next discoveries.

Validated hERG Cell Line for Cardiac Safety Screening

Manuel Pacheco — Tue, 01 Apr 2025 17:25:24 +0000

Explore how our CHO-K1 Kv11.1 (hERG) recombinant cell line was validated for use in Automated Patch Clamp (APC) electrophysiology using the SyncroPatch 384 from Nanion Technologies. This application note highlights high-throughput cardiac safety testing capabilities, pharmacological validation with known hERG inhibitors, and key electrophysiological properties

Naᵥ1.5 Brilliant Sodium assay

Manuel Pacheco — Tue, 07 May 2024 15:25:42 +0000

High-throughput Sodium Flux Assay for Naᵥ1.5 using Assay Ready Cells

Explore the potential of Voltage-gated sodium channels (Naᵥs) for treating various diseases using a new fluorescence-based assay. This post highlights a method for screening modulators targeting Naᵥ1.5, vital for cardiac function and targeted by antiarrhythmic drugs.

Cell-specific CRCs in co-cultures with Brilliant Sodium

MDD Investments — Mon, 12 Feb 2024 02:29:40 +0000

By combining Brilliant Sodium with automated image analysis, we calculate cell-specific potencies of ouabain, a Na⁺/K⁺-ATPase inhibitor, in a mixed co-culture of human embryonic kidney (HEK) and Chinese hamster ovarian (CHO) cells. Power your research with our Snapshot-compatible solutions – built for a more complex culture future.

Check out our Sodium Channel Assays by clicking here

Cell staining and experimental setup

HEK cells were stained with Cytotracker red prior to mixing with CHO cells for plating in a 96 well plate. The following day, cells were loaded with ING-2 AM (2011) using our Brilliant Sodium protocol (9000) for 1 h. Images were acquired at 5 minute intervals after the addition of varying ouabain concentrations using both GFP (for ING-2) and Texas Red (for Cytotracker red) filter cubes.

Ouabain is an inhibitor of the sodium/potassium-pump, which uses ATP to transport sodium and potassium against their concentration gradient to maintain a cell’s resting potential. When the pump is blocked, an increase in intracellular sodium is observed that can be measured using ING-2, a fluorescent sodium indicator. Different isoforms of Na⁺/K⁺-ATPase are known to have sensitivities to ouabain.

For general information on our Sodium Indicators, click here

Timelapse videos of cells treated with 167 μM Ouabain (separate channels)

All cell identification

All cells were identified as objects if they fit the following parameters in the GFP channel: Area > 15 um – 100 um, Intensity > 3000 RFUs.

Timelapse video of GFP channel with object overlay

HEK293 cell identification

HEK cells were identified in each frame of the video if they fit the following additional parameters: Texas Red intensity > 5000 RFUs. Objects identified as HEK cells are outlined below. These objects are then overlaid onto aligned GFP images for the analysis. The remainder of the cells captured in the GFP mask are qualified as CHO cells.

Timelapse videos of HEK cell object identification

Data processing

Mean cell fluorescence for each cell population is tracked over time. All data is then normalized to the mean cell fluorescence for that population from the first image, acquired just after the addition of ouabain.

Generating cell-specific concentration response curves

Normalized data points at 45 minutes are used to generate a dose response curve for each cell population. A non-linear fit is used to calculate the IC50 for each population. Unique potencies are identified for human and rodent Na⁺/K⁺-ATPase isoforms that aligns with published data.

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