1) What an Ussing Chamber Measures & Why

Goal. Quantify transepithelial transport and barrier integrity across a tissue or monolayer separating apical and basolateral solutions.

Primary electrical readouts

• PD (Potential Difference, mV): open-circuit voltage across the epithelium.

• I_sc (Short-Circuit Current, µA/cm²): net active ion transport when PD is clamped to 0 mV.

• TER / TEER (Ω, Ω·cm²): barrier tightness; higher values = tighter junctions.

  • TEER is area-normalized: TEER = (Rsample − Rblank) × Area

Flux readouts (chemical)

• Paracellular tracer flux (e.g., mannitol, FITC-dextran): tight-junction permeability.

• Transcellular flux (e.g., glucose, amino acids): transporter-mediated uptake.

• Apparent permeability (P_app) for a solute: Papp = (dQ/dt) / (A × C0)

Why this matters. These metrics underpin research in CFTR/ENaC physiology, IBD, celiac disease, diarrhea models, airway hydration, skin barrier, and drug absorption/toxicity.

2) The Physics: Making Sense of PD, Isc, and TEER

• Ohm’s Law (tissue): V = I × R.

  When voltage-clamped to 0 mV, the injected I_sc equals net electrogenic transport (e.g., CFTR-mediated Cl⁻ secretion minus ENaC Na⁺ absorption).

• Series resistances (solution + electrodes + bridges) sit outside the tissue. Good chamber design places voltage-sensing electrodes close to the tissue to minimize series drop.

• AC vs DC TEER.

  • DC pulse/step: inject a small current briefly; compute ΔV/ΔI.

  • AC impedance: a small sinusoid (e.g., 1 kHz) can isolate epithelial resistance and capacitance if supported by the electronics.

Corrections to apply

• Liquid junction potentials (LJP): match ionic strength; use KCl agar bridges.

• Offset correction: short the sensing electrodes in symmetric solutions; set baseline PD ≈ 0 mV before mounting tissue.

• Area normalization: always report µA/cm² (I_sc) and Ω·cm² (TEER) using the true aperture area.

3) Ussing Chamber Hardware: What Each Component Does

Chambers and Sliders (Apertures)

• Classic split-halves: maximal flexibility; more handling skill required.
• EasyMount: guided, fast, repeatable mounting; reduces tissue damage and variability.
• Aperture determines sensitivity and normalization (common: 0.071–0.64 cm²).
• Gaskets (thickness, compliance) control compression; over-tightening causes edge leaks or ischemia.

Electrodes & Bridges

• Ag/AgCl electrodes via agar–KCl bridges (e.g., 3 M KCl in 2–3% agar).
• Keep sensing electrodes close to tissue; current electrodes further out.
• Maintenance: re-chloride Ag wires periodically; replace cloudy or dehydrated bridges.

Perfusion, Gas, Temperature

• Gas lift with 95% O₂ / 5% CO₂ (carbogen) for bicarbonate buffers; gentle bubbling reduces unstirred layers.
• Perfusion (constant or interval) prevents depletion/accumulation; maintains set drug concentrations.
• Temperature: 37 °C with in-block or in-line heaters and calibrated probes; even 1–2 °C drift changes I_sc/TEER.

Clamps and DAQ

• Voltage–current clamps (single/multi-channel) measure PD, inject current, calculate I_sc/TEER automatically.
• Data acquisition: time-stamped events, protocol steps, and annotations improve reproducibility.

Looking for systems? See Ussing Chamber Systems and the Ussing Chamber Protocol.

4) Ussing Chamber Solutions & Recipes (working examples)

Verify with your IACUC/IRB/SOP. Buffer chemistry affects LJPs, transport driving forces, and viability.

Krebs–Ringer Bicarbonate (typical)

• NaCl 117 mM, KCl 4.7 mM, CaCl₂ 2.5 mM, MgSO₄ 1.2 mM, KH₂PO₄ 1.2 mM, NaHCO₃ 25 mM, Glucose 10 mM
• Gas with 95% O₂ / 5% CO₂, 37 °C

Mannitol/Glucose Osmotic Pair (symmetric osmolality)

• Serosal: glucose 10 mM; apical: mannitol 10 mM (or vice versa) to isolate transport asymmetries.

Common modulators

• Amiloride (apical) → ENaC block (↓ Na⁺ absorption)
• Forskolin/IBMX → ↑ cAMP → ↑ CFTR-mediated Cl⁻ secretion
• Bumetanide (basolateral) → NKCC1 block (limits Cl⁻ loading → ↓ secretion)
• CFTRinh-172 → inhibits CFTR currents

Tracer examples for flux

• Paracellular: mannitol, FITC-dextran (3–4 kDa)
• Transcellular: radiolabeled glucose, amino acids (observe safety protocols)

5) Ussing Chamber Protocol: Step-by-step SOP (field-tested)

A. Pre-run checks (10–20 min)

1. Electrode offset: immerse both sensing tips in the same buffer; adjust PD ≈ 0 mV.
2. Bridge health: gentle suction → free flow; no bubbles.
3. Temperature: stabilize chambers at 37 °C (±0.2 °C).
4. Solutions: pre-warm and gas; verify pH (7.3–7.4) and osmolality (±5 mOsm).
5. Blank TER: assemble with a gasket/no tissue if SOP calls for R_blank.

B. Tissue/Insert prep

• Native tissue: dissect carefully; open along mesenteric border; trim to aperture.
• Monolayers (e.g., Transwell): rinse, equilibrate 15–30 min; use adapter sliders if needed.

C. Mounting (2–5 min with EasyMount)

• Align tissue (mucosa → apical, serosa → basolateral).
• Compress to seal—no wrinkles or edge extrusion.
• Start perfusion/gas; wait 10–20 min for baseline stabilization.

D. Baseline acquisition

• Record PD, I_sc at steady state (drift < 1%/min).
• Measure TER via ΔI or AC routine; compute TEER.

E. Interventions (examples)

1. Amiloride (apical) → ↓ I_sc (blocks ENaC)
2. Forskolin/IBMX (bilateral or basolateral) → ↑ I_sc (activates CFTR)
3. Bumetanide (basolateral) → ↓ secretory current (blocks NKCC1)
4. Test compounds apical/serosal; track PD/I_sc over time; collect samples for flux.

F. Flux sampling

• Define intervals (e.g., every 10–15 min); maintain sink conditions (receiver < 10% of donor concentration).
• Calculate P_app and clearance; normalize to area and time.

G. Close-out & QC

• Recheck offset and blank to confirm stability.
• Log temperature/pH deviations; inspect tissue for edge damage.

6) Ussing Chamber Data Analysis, Normalization & Reporting

Normalization

• I_sc to µA/cm²: divide by aperture area.
• TEER to Ω·cm²: (Rsample − Rblank) × Area
• Report means ± SEM, n animals, n tissues/animal; avoid pseudo-replication.

Typical plots

• Time course of I_sc with annotated additions.
• TEER trajectory pre- and post-treatment.
• Flux vs time; bar charts of P_app.

Stats

• Paired analysis when the same tissue serves as its own control.
• Mixed models for multi-animal, multi-tissue designs.
• Pre-define exclusion criteria (leaks, unstable baselines).

7) Ussing Chamber Troubleshooting (fast diagnostics)

8) Experimental Design by Tissue Type (Ussing Chamber)

Intestine/Colon

• Consider indomethacin pre-incubation if prostaglandins confound responses.
• Gentle mucosal cleaning; avoid epithelial tears.

Airway (trachea/bronchus)

• ENaC block with amiloride (apical); CFTR activation with forskolin/IBMX; observe bumetanide sensitivity.

Skin

• Thicker, higher TEER; ensure robust sealing; allow longer equilibration.

Cultured Monolayers (e.g., Caco-2, primary airway)

• Verify confluence/TEER thresholds; mind filter support resistance (subtract blank); use adapter sliders.

9) Quality & Reproducibility Checklist

• Chamber model, aperture, gasket lot recorded
• Temperature/pH logs within range
• Electrode offset before/after
• Baseline stability (<1%/min drift)
• Correct side additions and timestamps
• Area normalization documented
• Exclusion criteria predefined and applied
• Raw files archived (time series + event log)

10) Safety & Compliance

• Radiotracers: licensing, shielding, wipe tests, segregated waste.
• Human/animal tissues: IRB/IACUC approvals, appropriate biosafety precautions.
• Chemical hazards: bumetanide, forskolin, DMSO controls; maintain SDS access.

11) Selecting & Budgeting the Right Ussing Chamber System

Match to goals

• Teaching / pilot work: single channel, manual clamp, basic perfusion.
• Core lab / pharma: multi-channel (4–8+), automated routines, integrated DAQ.
• Barrier screening: stable TEER measurement with rapid mounting (EasyMount), reliable perfusion & temperature control.

Cost expectations (configuration-dependent)

Cost expectations vary depending on several factors (number of channels and chamber set-ups). We configure systems by tissue, aperture, and throughput.

12) Ussing Chamber Appendices

A) Quick-start SOP (one page)

1. Warm/gas buffers; set 37 °C.
2. Zero electrodes in symmetric buffer.
3. Mount tissue (no wrinkles, correct orientation).
4. Stabilize; record baseline PD/I_sc; measure TER → TEER.
5. Add modulators (amiloride → forskolin/IBMX → bumetanide, etc.).
6. Collect flux samples on schedule; keep sink conditions.
7. Close-out: re-check offsets; document exclusions.

B) Common Calculations

• I_sc density: Isc (µA/cm²) = Iraw (µA) / Area (cm²)
• TEER: TEER (Ω·cm²) = (Rsample − Rblank) × Area
• P_app: Papp = (ΔQ/Δt) / (A × C0)
• Conductance: Gt = 1 / R (normalize to area as needed)

C) Reporting Template (copy/paste)

• Tissue: species/region; monolayer line & passage
• Chamber: model, aperture (cm²), gasket thickness
• Solutions: compositions, pH, temperature; gas mix
• Clamp: model, sampling rate, TEER method (DC/AC)
• Baseline PD/I_sc/TEER ± SEM (n animals, n tissues/animal)
• Agonists/antagonists: side, concentration, vehicle control
• Flux: tracer, sampling schedule, P_app calculation
• Statistics: tests, α level, exclusion criteria

13) Ussing Chamber FAQs