I am trying to do patch clamp (whole cell) electrophysiology on astrocytes in culture (namely, primary human fetal astrocytes). It seems for astrocytes, an external solution of pH 7.2 is often used, instead of pH 7.4. Is there a reason for this? this seems awfully acidic - but it may be helping me patch. Also, it seems people tend to use very high internal EGTA to remove Ca++ (e.g. papers I've seen use 5 and 10 mM). Is this needed? This could create a problem for us.
Usually the internal solution is acidic with a pH of 7.2 and the external has a pH of 7.4. This is because usually the intracellular pH is acidic compared to the extracellular fluid which has a pH of 7.4 in physiological conditions.
Did you mean acidic pH for the internal or the external?
EGTA is used in the internal solution for various reasons depending on the current of interest. Recording of calcium currents require EGTA or BAPTA (preferred) to prevent calcium induced inactivation of calcium currents. Calcium chelators are also used to prevent activation of calcium activated potassium channels.
Which currents are you planning to record?
Thanks for the information. I am aware that external pH is typically 7.4. However, for cultured *astrocytes*, peoples use EXTernal pH of 7.2, not 7.4. This is what is confusing to me. this seems quite acidic for an external solution. The astrocytes we are studying are relatively unstudied - so we'd first characterize the channels on them. Probably K+ and maybe calcium. If they had sodium current and action potentials, that would be quite interesting. but, i'm doubting that right now.
I've experience in patching cultured astrocytes (Podda MV et al, Glia 2012) obtained from brain cortex and never had problems with standard internal or external solution commonly used for neurons. Astrocytes tend to be quite flat in culture and therefore you have less probability to do good seals with "the first shot".
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