When using electrophysiology techniques such as patch-clamp recording, all the solutions and equipment coming into contact with the cells must be sterile and of the highest purity available. Water is used for rinsing and to prepare the buffered solutions used to mimic the physiological conditions the cells were accustomed to (the cell culture media in the case of cultured cells). An extracellular solution (ECS) is used to bathe the cells, and an intracellular solution (ICS) is used to fill the pipettes. The ionic composition, pH and osmolarity of these solutions must be carefully controlled.
If brain slices are used, artificial cerebrospinal fluid (ACSF) or a similar solution is used in order to mimic in vivo conditions. This solution is used both to prepare the brain slice and to perfuse it during the experiment. It may also be used as the extracellular solution during patch clamp measurement. The ACSF solution must be freshly prepared and pH, osmolarity and ionic composition must also be carefully controlled. The water used to prepare the ACSF solution must be free of compounds that may affect the tissue or interfere with the experiment.
The following water contaminants should be avoided:
Ions Since the osmotic pressure of the solutions must be carefully controlled, the water used to prepare the solutions should contain the least amount possible of ions. In addition, many ion channels are sensitive to ions, especially cations, which should be controlled. Water with a resistivity of 18.2 MΩ.cm ensures very low concentration of ionic species in the water.
Heavy metals Heavy metals may affect cell function. In addition, many ion channels are sensitive to heavy metals, in particular Calcium channels. Therefore the water used for ion channel studies must be free of heavy metals.
Bacteria and endotoxins Bacteria can release a variety of ions and organic compounds into the solution. Bacteria contamination can also cause sudden changes in pH. In addition, most Gram-negative bacteria release endotoxin (pyrogenic substance), a complex lipopolysaccharide (LPS). Endotoxins may lead to changes in cell growth and function. The use of ultrafiltration ensures endotoxin-free water and bacteria levels below 10 cfu/100mL.
Organic compounds Small organic molecules are commonly present in raw water supplies (humic acids, tannins, pesticides, endocrine disrupters, etc.) and may remain in tap water. They may affect cell function, and affect experimental results. A low TOC value ensures low concentrations of plasticizers and other organic molecules in the water.
In conclusion, using freshly purified ultrapure water is recommended for electrophysiology experiments. Using an ultrafiltration cartridge (BioPak®) at the point of use of the water purification system eliminates the risk of endotoxin contamination.
Many laboratories performing these experiments also culture cells. For more information on the importance of water for cell culture, please see: cell culture.