I am recording the sodium currents in whole cell patch clamp methods in DRG neurons (20 micrometer) from the Axopatch-200B and getting capacitance 1.24 pF in the dial. Should the capacitance be divided by the size of the cell or not as it is normally 1 microfarad/cm2?
Dear Dr. Singh,
The WHOLE CELL CAP dial of Axopatch 200B gives you an absolute measure of the cell capacitance. In order to get the measurement of specific membrane capacitance, the measurement from the WHOLE CELL CAP dial should be divided by the surface area of the cell. Assuming the diameter (D) of the DRG neuron is 20 um, the surface area is 1256.64 um^2 or 1.26 x 10^-5 cm^2 (area = 4 x pi x r^2 or pi x D^2). However, your measurement of the absolute capacitance seems too small than usually reported for the DRG neurons (Holohean et al, Brain Research 711 1996: 138-145) . Is it 12.4 pF and not 1.24 pF? I f so, dividing the measured cell capacitance value (12.4 pF, or 12.4 x 10^-6 uF) by the calculated area (1.26 x 10^-5 cm^2) will give you the value of specific membrane capacitance of 0.98 uF/cm^2. More details could be found in Gentet et al, Biophysical Journal 79 2000: 314-320.
Anton.
Dear Dr. Singh,
The WHOLE CELL CAP dial of Axopatch 200B gives you an absolute measure of the cell capacitance. In order to get the measurement of specific membrane capacitance, the measurement from the WHOLE CELL CAP dial should be divided by the surface area of the cell. Assuming the diameter (D) of the DRG neuron is 20 um, the surface area is 1256.64 um^2 or 1.26 x 10^-5 cm^2 (area = 4 x pi x r^2 or pi x D^2). However, your measurement of the absolute capacitance seems too small than usually reported for the DRG neurons (Holohean et al, Brain Research 711 1996: 138-145) . Is it 12.4 pF and not 1.24 pF? I f so, dividing the measured cell capacitance value (12.4 pF, or 12.4 x 10^-6 uF) by the calculated area (1.26 x 10^-5 cm^2) will give you the value of specific membrane capacitance of 0.98 uF/cm^2. More details could be found in Gentet et al, Biophysical Journal 79 2000: 314-320.
Anton.
What Software are you using for recording? Usually pClamp give you automatically the value of cell capacitance and can be also recorded. Of course once you have the total cell capacitance you need to divide for membrane area to get the specific capacitance. In pClamp, once got the whole cell configuration, if you open in menu "Membrane Test" you have visible "Cell Capacitance", Membrane Resistance, Serie Resistance and value of Tau.
I hope this can be useful for you
i am not sure what Anton is saying above with regards to specific vs absolute capacitance but on all axon amplifiers I have ever used, the number on the dial IS the cell capacitance (no need to divide). If you want to get to cell size from the capacitance, that's another story.
The number you are getting sounds way too small. If your correction is incorrect then you can't trust that number.
You can measure it yourself with a small depolarizing step in voltage clamp (~5 mV from holding) by fitting the decay of the current. There are other ways using area-under-the-curve of the transient as well.
Since the value of 1 microfarad/cm2 was mentioned, I was thinking that Jitendra is asking about the value of specific capacitance (in uF/cm^2) and why he is not getting 1 uF/cm^2 which is normally considered. The so called specific capacitance is just a conversion factor between the measured absolute capacitance, or capacitance of the entire cell membrane, and the cell surface area. Usually, the capacitance of 1 cm^2 of the cell membrane is about 1 uF. My calculation above shows that the measured value of cell (absolute) capacitance is too small. And yes, I agree with Oliver that there are better ways of measuring the capacitance than reading it from the dial. The paper of Gentet et al. in Biophysical Journal describes these measurements.
I see what you mean Anton. But going back to Jitendra's issue, I was thinking he might have a wrong unit. This depends on the Beta value for his headstage.
The number could well be 124 pF or maybe 1.24 nF.
After going whole cell following correction of pipette capacitance you can give a 10 mV step to record the cell capacitance transient. Integrate the area under curve of cell capacitance and divide by 10 mV to get the whole cell capacitance in pF. Statistics function in clampfit can be used to measure the area under the capacitance transient.
The dial for whole-cell capacitance is 10 pF (/beta) per turn, so if beta equals 1, a value of 1.24 on the dial would equal 12.4 pF capacitance. That may account for the error mentioned by Dr. Sheinin.
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