Whole-cell tools
Ephys solution calculator
EPSC Internal (low chloride)
Cs⁺/methanesulfonate, low-chloride formulation for isolating excitatory synaptic currents. Evidence & sources →
| Compound | Final mM | MW g/mol | Source | Stock mM | Add | Remove |
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Low-chloride EPSC internal solution
This cesium-based internal is intended for voltage-clamp recordings of excitatory postsynaptic currents. Intracellular chloride influences the chloride reversal potential. Interpret current polarity using the holding potential, external solution, and internal composition; the internal recipe alone does not isolate a receptor-mediated current.
Hamill et al. (1981): whole-cell patch-clamp methodsWhy ATP and GTP are included
ATP supports energy-dependent cellular processes, while GTP supports G-protein signaling. Check the exact nucleotide salt, hydration state, and assay on the product label before weighing. The molecular weight must match the reagent used.
When should I measure pH?
Check final pH after every ingredient, including ATP and GTP, has dissolved. Equilibrate to the protocol’s specified measurement temperature and record it. Nucleotide additions can change pH; the size of the change depends on the salt forms and buffering.
HEPES-buffered solutions become more alkaline when cooled. A HEPES-only estimate using −0.014 pH units per °C gives an increase of about 0.29 when cooling from 22 °C to 1 °C. This is an approximation, not a correction to apply automatically to a complete internal solution.
HEPES temperature coefficient: manufacturer technical sheetDo ATP and GTP require preparation on ice?
Cold storage guidance does not establish a universal requirement to perform every preparation step on ice. Use the handling conditions validated for your nucleotide products and complete internal formulation. The stability of a complete solution during an hour at room temperature should not be assumed from powder shipping conditions. Keep final pH measurement temperature separate from storage and aliquoting temperature.
Mg-ATP handling and solution stability: manufacturer technical sheetInternal solution recipe
EPSC Internal (low chloride)
Voltage clamp · EPSC
| Compound | Final (mM) | MW (g/mol) | Source | Stock (mM) | Add |
|---|---|---|---|---|---|
| Cs-methanesulfonate | 135 | 228.00 | Powder | — | 1539.0 mg |
| HEPES | 10 | 238.30 | Stock solution | 100 | 5000 µL |
| EGTA | 1 | 380.35 | Stock solution | 100 | 500 µL |
| MgCl₂ | 1 | 203.30 | Stock solution | 1,000 | 50 µL |
| Mg-ATP | 4 | 529.47 | Powder | — | 105.9 mg |
| Na-GTP | 0.3 | 567.10 | Powder | — | 8.5 mg |
| Na₂-phosphocreatine | 8 | 255.10 | Powder | — | 102.0 mg |
Internal Solution Preparation
- Add approximately 35–40 mL of room-temperature ultrapure water to a 50 mL conical tube.
- Add the main salt and mix until completely dissolved.
- Pipette in the required stock solutions and mix thoroughly.
- Weigh and add ATP, GTP, and Na₂-phosphocreatine. Mix until all components are completely dissolved.
- Adjust the pH to the value specified in the recipe: use CsOH for Cs⁺-based voltage-clamp internals and KOH for the K-gluconate current-clamp internal.
- Bring the solution close to its intended final volume, then measure and adjust its osmolality. Bring the solution to the exact final volume of 50 mL.
- Pass the completed solution through a sterile 0.22 µm filter.
- Place the filtered solution on ice and divide it into single-use aliquots.
- Store the aliquots at −80 °C. Avoid repeated freeze–thaw cycles.