Instructions for Using the CFE

The ProCFE is a polypropylene-insulated carbon-fiber microelectrode used in the detection and localization of oxidizable or reducible analytes. It is sufficiently noise-free and fast to record quantal secretion from single cells.

1. Detectable substances: Oxidizable neurotransmitters include catecholamines (epinephrine, norepinephrine, dopamine), indolamines (5-HT) and their metabolites. Small peptides containing tryptophan, tyrosine, or cysteine may also be detectable. Usually, the ProCFE in a voltage range of 600-900 mV oxidizes these compounds.

2. Setting up to use the ProCFE: You will need a voltage clamp amplifier (also referred to as a potentiostat) which is capable of clamping the voltage to a minimum of 600 mV. Other special working conditions for the ProCFE include:

  • making the electrical connection with the carbon-fiber electrode;
  • mounting the ProCFE on the voltage clamp amplifier (potentiostat); and
  • applying an appropriate detection potential

Electrical contact with the carbon fiber is made by back-filling the electrode with a conducive liquid (e.g., KCl or Hg) which is then placed in contact with the input wire of a preamplifier (headstage). The back-filling liquid for the ProCFE (ca. 10 uL) can be either KCl (0.2-3 M), NaCl (<4M) or mercury. From, a performance aspect, KCl or NaCl is less desirable than mercury because the electrode noise of KCl or NaCl -filled ProCFEs has been observed to increase several hours after back filling. This makes it impossible to reuse the electrodes. To connect the electrode to the headstage you may use a standard patch-clamp pipette holder with the small adapter provided. (See figure 1 and 2 for mounting and back filling the ProCFE). A chlorided silver wire is needed for a stable electrical connection when KCl or NaCl is used as the back filling solution. For mercury, chloriding the silver wire is not required. The mercury can be reused with each electrode (figure 2). (Do not discard Hg as trash, It is considered hazardous material.)

The appropriate applied voltage usually exceeds the redox potential of the species being investigated. For example, the redox potential for catecholamines is approximately 100-200 mV under physiological conditions. In practice, 600-900 mV is usually employed for amperometry to overcome the effects of electrochemical kinetics. The potential can sometimes be set directly on the instrument; or a DC voltage can be applied to the voltage command input of your patch-clamp amplifier.

3. Testing the ProCFE: A good ProCFE should add less than 1pAp-p to your overall system noise when the electrode is placed in contact with the buffer solution (30-500 Hz bandwidth). However, some increase in noise is expected. In normal cases, the systems noise increases 1-5 times after the electrode enters the bath solution. No increase in noise upon the electrode placement indicates the lack of an electrical connection to the carbon-fiber surface. Too much noise indicates a broken tip or insufficient insulation of the carbon fiber.

The electrode response can be checked with a locally applied test solution of catecholamines (1-10 uM). The electrode is fixed at 700 mV in the buffer solution. The test solution is loaded into micropipette and pressure ejected onto the electrode surface. If the electrode is functioning properly a positive current response of approximately 1-2pA/uM will be observed. Strong convection caused by fluid flowing to the electrode surface is likely to increase the observed current.

4. Application of the ProCFE: The ProCFE can be used with any cells or tissues that secrete oxidizable substances (e.g. catecholamines or indolamines). To detect individual quantal release. The cell surface facing ProCFE must be clean. Thus culture cells are particularly good for high sensitivity recording. Typical cells include adrenal chromaffin cells, mast cells and PC12 cells. Place the ProCFE tip directly adjacent to the cell surface or gently touch the cell then stimulate (e.g., apply a secretagogue such as potassium to the cell).

 A used ProCFE tip may still detect secretion by individual vesicles, but if the tip is contaminated (for example, after touching and leaving the cell surface many times) the sensitivity of the ProCFE will decline. The signals will appear lower in amplitude and appear slower than those observed at fresh electrodes. For the most sensitive recordings, each ProCFE should only be used with five cells.

 References:

  1. Wightman, et al. Proc. Natl. Acad. Sci. 88:10754-58, 1991.
  1. Chow, et al. Nature. 356:60-63, 1992.
  2. Alvarez de Toledo, et al. Nature. 363:554-558, 1993.
  1. Zhou, Z and Misler, S. J. Biol. Chem. 270:3498-3505, 1995.
  2. Zhou, Z and Misler, S. Chow, R.H. Biophys. J. 70:1542-1552, 1996.
  3. Zhou, Z and Misler, S. J. Biol. Chem. 271: 270-271, 1996.
  4. Zhou, Z and Misler, S. Proc. Natl. Acad. Sci. 92: 6938-6942, 1995.
  5. Kawagoe, et al. J. Neurosci. Method 48:225-240, 1993.
  6. Chow, R.H., von Ruden, L. In: Sakmann, B. and Neher, E., Eds. Single Channel Recording (2nd edition). Plenum Press, New York, 1995.
  7. Stamford, et al. In: Stamford, J.A., Ed Monitoring Neuronal Activity. IRL Press, Oxford. Pp:113-146, 1992.
  8. Zhuan Zhou. Restoring CFE Sensitivity by a simple cutter. AxonBits, 18:5, 1996..

 

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Warning: This equipment is not intended for use in human applications or human experimentation.

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