Whole-cell tools
Ephys solution calculator
IPSC Internal (high chloride)
CsCl-based, high-chloride formulation for sensitive inhibitory-current detection. Evidence & sources →
| Compound | Final mM | MW g/mol | Source | Stock mM | Add | Remove |
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High-chloride IPSC internal solution
This high-chloride internal changes the chloride driving force for inhibitory postsynaptic current recordings. The expected current direction depends on the holding potential and external chloride as well as the internal solution. Keep these conditions in the recording record when comparing IPSCs.
Coombs et al. (1955): ionic mechanisms of inhibitionWhy 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
IPSC Internal (high chloride)
Voltage clamp · IPSC
| Compound | Final (mM) | MW (g/mol) | Source | Stock (mM) | Add |
|---|---|---|---|---|---|
| CsCl | 140 | 168.40 | Powder | — | 1178.8 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.