Custom Assay Development · Phospho Flow

Track phosphorylation signaling in your clinical samples

Start with Teiko's validated 33- or 41-marker mass cytometry panel and add up to 10 custom markers to measure target engagement, pathway activation, and pharmacodynamic response. Track phospho-signaling directly with readouts like pSTAT3, pERK1/2, and pS6.

Start a project How we capture phospho signals →

THE PROCESS

Select target phospho markers

Run each sample on a fit-for-purpose cytometry panel that can profile 840+ immune cell populations.

Add custom phospho markers

Design a FFP panel around your specific target, pathway, or mechanism of action.

View your results

Get reproducible pharmacodynamic insights without building and validating a panel yourself.

How Teiko Does It

Phospho Flow Panel Development

From selecting targets to a validated phospho flow panel.

You provide

Define targets & sample type

Identify the phospho markers to include and decide whether the panel will be built for whole blood or PBMCs.

Teiko builds

Select clones & run titrations

Choose the antibody clones and reagents needed, then test concentrations to determine the staining conditions that give the best signal quality.

Teiko evaluates

Permeabilization conditions

Test permeabilization conditions to get clean signal with strong cell recovery.

Teiko establishes

Stimulation conditions

Compare stimulated and unstimulated samples.

pSTAT3 Validation →

Teiko delivers

Panel & readout strategy

Bring the validated markers and conditions together into a finished panel and define how the data will be displayed and interpreted.

Check out our assay validation report →

pSTAT3 Validation

Measuring STAT3 and pSTAT3 in a TokuKit-fixed blood sample

STAT3 is phosphorylated when a cell is actively signaling. At that point, STAT3 becomes pSTAT3. By stimulating with IL-6, we activate the pathway, providing a signal for detection. We then use TokuKit to stabilize the signal and measure pSTAT3 levels. When creating your fit-for-purpose panel, we apply this method for each marker, identifying the appropriate stimulation conditions required to activate each one.

Our internal validation protocol

Prepare IL-6

Collect whole blood into a vacutainer.

Stimulation with IL-6, incubate 15 mins at 37°C.

Fix sample with TokuKit: move blood into Buffer 1 for 15 minutes, then transfer the entire mixture into Buffer 2.

Store fixed samples at −80°C until analysis.

Capture phospho state at collection to prevent degradation

TokuKit fixes samples at the point of collection, locking in the phospho proteins before shipping or processing can change them, so they're ready for cytometry analysis. Check out our TokuKit validation studies on mass and spectral, including a list of 120+ validated markers and clones.

See validated markers & stability data →

STAT3 & pSTAT3 histogram overlays: unstimulated vs. IL-6 stimulated whole blood, TokuKit-fixed

STAT3 and pSTAT3 histogram overlays for unstimulated and IL-6 stimulated whole blood

pSTAT3+ gating: total STAT3 is unchanged, pSTAT3 increases with IL-6 stimulation

STAT3 and pSTAT3 gating plots for unstimulated and IL-6 stimulated whole blood

TokuKit-validated clones for phospho flow

On the dashboard

Visualize immune changes across markers, populations, and drugs.

Once your panel is built, we run your samples and track total and phospho protein levels across your study endpoints. Timepoints shown here are just one example, all visualized on your dashboard, interactive and shareable.

pSTAT3 by timepoint

CD4+ T cell pSTAT3 signal across a study, one box per timepoint.

pSTAT3 expression by timepoint box and whisker chart

pSTAT3 marker expression heatmap

Shows median channel value (MCV) for each marker on each cell type. Higher values indicate stronger expression.

Why it matters

Phospho flow informs clinical decisions.

Phospho flow supports many translational, biomarker, and clinical questions. Here are three ways teams put it to work.

Predict treatment response

Comparing pathway activation before and after treatment gives an early signal of who is and isn't responding, well before clinical outcomes play out.

Screen mechanism of action

By measuring the exact signaling step a drug is meant to block, you can confirm it's working as intended and compare candidate compounds side by side.

Track disease status

Changes in phospho signal can track disease status over time, giving a clear readout of whether a patient is moving toward remission.

Good to know

Frequently asked

Basics

Why measure phospho signal instead of total protein?

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Total protein expression only tells you how much of a protein is present, not whether it's active. Phospho flow measures the phosphorylated form directly, showing whether a signaling pathway has actually been switched on, the readout that matters for target engagement and pathway activation.

Platform

What instrument is used to run the assay?

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Panels are built and validated using mass cytometry.

Stimulation

How do you know which stimulus to use for each marker?

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Each phospho target has a specific stimulus, for example IL-6 for STAT3 or PMA for S6. We select and validate the stimulation condition for your specific target before building it into the panel.

TokuKit

Which clones and markers are validated on TokuKit?

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We've validated over 120 antibody clones and markers on TokuKit-fixed samples for mass and spectral cytometry, including phospho targets like STAT3 and pSTAT3. Visit teiko-labs.com/tokukit →

Whole blood

Why measure phospho signal in whole blood instead of PBMCs?

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Preserved whole blood captures the phospho signal as it actually was in the patient at the moment of the blood draw, rather than a signal distorted by isolation, freezing, and ex-vivo re-stimulation. It also lets you batch an entire patient time series and run every sample together, removing batch-to-batch variation from the readout.