Improved superfusion technique for rapid cooling or heating of cultured cells under patch-clamp conditions.
We have developed an improved technique for fast cooling and heating of solutions superfusing isolated cells under patch-clamp or calcium imaging conditions. The system meets the requirements for studying temperature dependency of all kinds of ion channels, in particular temperature-gated ion channels. It allows the application of temperature changes within a range of 5-60 degrees C at maximum rates of -40 degrees C/s to 60 degrees C/s. Barrels filled with different solutions are connected to a manifold consisting of seven silica capillaries (320 microm inner diameter, i.d.). A common outlet consists of a glass capillary through which the solutions are applied onto the cell surface. The upper part of this capillary is embedded in a temperature exchanger driven by a miniature Peltier device which preconditions the temperature of the passing solution. The lower part of the capillary carries an insulated copper wire, densely coiled over a length of 7 mm, and connected to a dc current source for resistive heating. The Peltier device and the heating element are electrically connected to the headstage probe which is fixed on to a micromanipulator for positioning of the manifold. The temperature of the flowing solution is measured by a miniature thermocouple inserted into the common outlet capillary near to its orifice which is placed at a distance of less than 100 microm from the surface of the examined cell. The temperature is either manually controlled by voltage commands or adjusted via the digital-to-analog converter of a conventional data acquisition interface. Examples are given of using the device in patch-clamp studies on heterologously expressed TRPV1, TRPM8, and on cultured rat sensory neurons.
Dittert
Ivan
I
Department of Cellular Neurophysiology, Institute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic.
Benedikt
Jan
J
Vyklický
Ladislav
L
Zimmermann
Katharina
K
Reeh
Peter W
PW
Vlachová
Viktorie
V
D000200:Action Potentials / Q000502:physiology*; D000818:Animals; D018929:Cell Culture Techniques / Q000295:instrumentation* / Q000379:methods; D002478:Cells, Cultured; D003080:Cold Temperature; D004780:Environment, Controlled; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006358:Hot Temperature; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology*; D009475:Neurons, Afferent / Q000502:physiology; D018408:Patch-Clamp Techniques / Q000295:instrumentation* / Q000379:methods; D010477:Perfusion / Q000295:instrumentation*; D051381:Rats