Ussing Chamber Setup & Calibration: TEER, Isc, and CFTR Assay Basics (with Troubleshooting)
This guide outlines a complete, repeatable Ussing workflow—from assembling and calibrating your chamber to executing a canonical CFTR assay and calculating TEER and Isc with precision. Designed for epithelial transport researchers, this step-by-step reference helps minimize noise, improve reproducibility, and ensure consistent, publication-quality results.
What the Ussing Chamber Measures
- Potential Difference (PD): The transepithelial voltage (mV) across the tissue or monolayer.
- Short-Circuit Current (Isc): Net ion transport measured when PD is clamped to 0 mV.
- Transepithelial Electrical Resistance (TEER): Barrier integrity measured from the applied perturbation and resultant current (Ω·cm² after area normalization).
When assessing barrier function (tight junctions), focus on TEER. When evaluating ion channel or CFTR activity, prioritize Isc under voltage clamp control.
Setup And Calibration Checklist
- Chamber assembly: Inspect gaskets, O-rings, and aperture surfaces. Proper sealing ensures stable baselines. A modern Ussing chamber system like the EasyMount P2300 simplifies alignment and leak prevention.
- Solutions: Use freshly prepared, pH-balanced buffers. Pre-warm and maintain osmolarity across both sides.
- Electrodes & bridges: Condition Ag/AgCl electrodes and refill agar bridges (typically 3 M KCl). Air bubbles cause drift—remove them carefully.
- Zeroing & offset: With no tissue mounted, verify PD ≈ 0 mV. After mounting, clamp to 0 mV and confirm baseline stability before adding reagents.
- Leak & bubble check: Tap gently to dislodge bubbles; re-seat gaskets if instability persists.
- Acquisition & logging: Record at 2–10 Hz and annotate each reagent addition. Use data acquisition software to automate normalization and graph export.
Canonical CFTR Assay Sequence
For airway or intestinal epithelia, a typical pharmacological sequence is:
- Amiloride (apical): Inhibits ENaC — reduces Na⁺ absorption (decrease in Isc).
- Forskolin + IBMX: Elevates cAMP — activates CFTR (increase in Isc).
- CFTR(inh)-172: Blocks CFTR — confirms CFTR-specific current (Isc drops back).
Consistency is key: maintain reagent concentration, timing, and side of addition across experiments.
Calculations & Normalization
Isc Normalization
Isc,norm (µA/cm²) = Isc,raw (µA) ÷ Area (cm²)
Example: 18 µA across a 0.33 cm² aperture = 54.5 µA/cm².
TEER Conversion
TEER (Ω·cm²) = Rmeasured (Ω) × Area (cm²) − Rblank (Ω·cm²)
Always subtract the blank resistance from an insert without cells, recorded under identical conditions.
Choosing the Right Hardware
For reproducible results, pair a precision voltage/current clamp amplifier with a durable, easy-to-clean Ussing chamber system. Add data acquisition software to complete the workflow — enabling seamless event logging, area normalization, and data export for analysis.
Fast Troubleshooting Guide
| Symptom | Likely Cause | Recommended Fix |
|---|---|---|
| Drifting baseline / noisy PD | Unconditioned electrodes, bubbles, or dirty salt bridges | Recondition electrodes; refill bridges; purge air bubbles |
| Asymmetric currents | Unequal buffer osmolarity or electrode imbalance | Recheck buffer prep; confirm identical salt bridges |
| Weak Isc response | Low tissue viability or damaged monolayer | Reduce handling time; verify cell confluence; replace tissue |
| TEER values fluctuate | Temperature instability or chamber leak | Stabilize thermal conditions; confirm seal integrity |
| Flat response after Forskolin | Expired reagents or poor CFTR expression | Prepare fresh Forskolin/IBMX; verify culture conditions |
Tip: Always document solution composition, electrode history, and tissue source. These metadata drastically improve reproducibility when re-analyzing data months later.
Ready to standardize your Ussing runs? Explore the EasyMount Ussing Chamber and VCC MC8 Voltage Clamp for stable, repeatable epithelial transport data.