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Dynamics of T-Junction Solution Switching Aimed at Patch Clamp Experiments.

Auzmendi JA, Smoler M, Moffatt L - PLoS ONE (2015)

Bottom Line: The exchange time was found to increase quadratically with the delay, although a sizeable variability remains unexplained by this relationship.This effect would be present in all tubing based devices.Present results might be of fundamental importance for the adequate design of serial compound exchangers which would be instrumental in the discovery of drugs that modulate the action of the physiological agonists of ion channels with the purpose of fine tuning their physiology.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Química Física de los Materiales, Medio Ambiente y Energía. Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.

ABSTRACT
Solutions exchange systems are responsible for the timing of drug application on patch clamp experiments. There are two basic strategies for generating a solution exchange. When slow exchanges are bearable, it is easier to perform the exchange inside the tubing system upstream of the exit port. On the other hand, fast, reproducible, exchanges are usually performed downstream of the exit port. As both strategies are combinable, increasing the performance of upstream exchanges is desirable. We designed a simple method for manufacturing T-junctions (300 μm I.D.) and we measured the time profile of exchange of two saline solutions using a patch pipette with an open tip. Three factors were found to determine the timing of the solution switching: pressure, travelled distance and off-center distance. A linear relationship between the time delay and the travelled distance was found for each tested pressure, showing its dependence to the fluid velocity, which increased with pressure. The exchange time was found to increase quadratically with the delay, although a sizeable variability remains unexplained by this relationship. The delay and exchange times increased as the recording pipette moved away from the center of the stream. Those increases became dramatic as the pipette was moved close to the stream borders. Mass transport along the travelled distance between the slow fluid at the border and the fast fluid at the center seems to contribute to the time course of the solution exchange. This effect would be present in all tubing based devices. Present results might be of fundamental importance for the adequate design of serial compound exchangers which would be instrumental in the discovery of drugs that modulate the action of the physiological agonists of ion channels with the purpose of fine tuning their physiology.

No MeSH data available.


Related in: MedlinePlus

T-junction manufacture and operation.(A) Silicone tubing and punch. A 0.5 mm interval ruler is apparent on the bottom. (B) A hole is drilled perpendicular to the central hole. (C) After inserting a polyethylene tubing the T-junction is completed. (D) General device. I. N2 tank. II. Filter III. Pressurized reservoir of solution. IV. 3-way solenoid pinch valves. V. A 24 V custom made valve driver. VI. Merger system of two T-junctions staked in XYZ translator. VII. Open tip recording pipette. VIII. Patch clamp amplifier. IX. A multifunction data acquisition connected to PC. (E) Schematic representation of experimental configuration. (F) Image of experimental configuration. The image was taken using a web camera coupled to the setup microscope. (G) Operation of the solution switching. A pulse of test solution (violet) was inserted into the control solution (pink).
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pone.0133187.g001: T-junction manufacture and operation.(A) Silicone tubing and punch. A 0.5 mm interval ruler is apparent on the bottom. (B) A hole is drilled perpendicular to the central hole. (C) After inserting a polyethylene tubing the T-junction is completed. (D) General device. I. N2 tank. II. Filter III. Pressurized reservoir of solution. IV. 3-way solenoid pinch valves. V. A 24 V custom made valve driver. VI. Merger system of two T-junctions staked in XYZ translator. VII. Open tip recording pipette. VIII. Patch clamp amplifier. IX. A multifunction data acquisition connected to PC. (E) Schematic representation of experimental configuration. (F) Image of experimental configuration. The image was taken using a web camera coupled to the setup microscope. (G) Operation of the solution switching. A pulse of test solution (violet) was inserted into the control solution (pink).

Mentions: T-junctions were built on 0.30 mm ID (Altec Products LtdBude, United Kingdom) silicone tubing (ST). A punch (Fig 1A) was made out of a 23 G syringe by cutting and filing the tip and grinding the blade with an Arkansas stone. The ST was drilled with the punch perpendicular to the central hole (Fig 1B); to prevent the collapse of the drilled holes, pressurized N2 was injected from one end of the tubing while the other end was sealed. Silicone residues were removed with forceps. After all perforations were made, a piece of 0.28 mm ID polyethylene tubing (PE-10 Intramedic, Becton Dickinson) was placed in each one of the punctured holes using a fine-tipped forceps to form T- connectors (Fig 1C). All the T-junctions were secured with epoxy glue (Poxipol, Acapol, Buenos Aires, Argentina).


Dynamics of T-Junction Solution Switching Aimed at Patch Clamp Experiments.

Auzmendi JA, Smoler M, Moffatt L - PLoS ONE (2015)

T-junction manufacture and operation.(A) Silicone tubing and punch. A 0.5 mm interval ruler is apparent on the bottom. (B) A hole is drilled perpendicular to the central hole. (C) After inserting a polyethylene tubing the T-junction is completed. (D) General device. I. N2 tank. II. Filter III. Pressurized reservoir of solution. IV. 3-way solenoid pinch valves. V. A 24 V custom made valve driver. VI. Merger system of two T-junctions staked in XYZ translator. VII. Open tip recording pipette. VIII. Patch clamp amplifier. IX. A multifunction data acquisition connected to PC. (E) Schematic representation of experimental configuration. (F) Image of experimental configuration. The image was taken using a web camera coupled to the setup microscope. (G) Operation of the solution switching. A pulse of test solution (violet) was inserted into the control solution (pink).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4503516&req=5

pone.0133187.g001: T-junction manufacture and operation.(A) Silicone tubing and punch. A 0.5 mm interval ruler is apparent on the bottom. (B) A hole is drilled perpendicular to the central hole. (C) After inserting a polyethylene tubing the T-junction is completed. (D) General device. I. N2 tank. II. Filter III. Pressurized reservoir of solution. IV. 3-way solenoid pinch valves. V. A 24 V custom made valve driver. VI. Merger system of two T-junctions staked in XYZ translator. VII. Open tip recording pipette. VIII. Patch clamp amplifier. IX. A multifunction data acquisition connected to PC. (E) Schematic representation of experimental configuration. (F) Image of experimental configuration. The image was taken using a web camera coupled to the setup microscope. (G) Operation of the solution switching. A pulse of test solution (violet) was inserted into the control solution (pink).
Mentions: T-junctions were built on 0.30 mm ID (Altec Products LtdBude, United Kingdom) silicone tubing (ST). A punch (Fig 1A) was made out of a 23 G syringe by cutting and filing the tip and grinding the blade with an Arkansas stone. The ST was drilled with the punch perpendicular to the central hole (Fig 1B); to prevent the collapse of the drilled holes, pressurized N2 was injected from one end of the tubing while the other end was sealed. Silicone residues were removed with forceps. After all perforations were made, a piece of 0.28 mm ID polyethylene tubing (PE-10 Intramedic, Becton Dickinson) was placed in each one of the punctured holes using a fine-tipped forceps to form T- connectors (Fig 1C). All the T-junctions were secured with epoxy glue (Poxipol, Acapol, Buenos Aires, Argentina).

Bottom Line: The exchange time was found to increase quadratically with the delay, although a sizeable variability remains unexplained by this relationship.This effect would be present in all tubing based devices.Present results might be of fundamental importance for the adequate design of serial compound exchangers which would be instrumental in the discovery of drugs that modulate the action of the physiological agonists of ion channels with the purpose of fine tuning their physiology.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Química Física de los Materiales, Medio Ambiente y Energía. Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.

ABSTRACT
Solutions exchange systems are responsible for the timing of drug application on patch clamp experiments. There are two basic strategies for generating a solution exchange. When slow exchanges are bearable, it is easier to perform the exchange inside the tubing system upstream of the exit port. On the other hand, fast, reproducible, exchanges are usually performed downstream of the exit port. As both strategies are combinable, increasing the performance of upstream exchanges is desirable. We designed a simple method for manufacturing T-junctions (300 μm I.D.) and we measured the time profile of exchange of two saline solutions using a patch pipette with an open tip. Three factors were found to determine the timing of the solution switching: pressure, travelled distance and off-center distance. A linear relationship between the time delay and the travelled distance was found for each tested pressure, showing its dependence to the fluid velocity, which increased with pressure. The exchange time was found to increase quadratically with the delay, although a sizeable variability remains unexplained by this relationship. The delay and exchange times increased as the recording pipette moved away from the center of the stream. Those increases became dramatic as the pipette was moved close to the stream borders. Mass transport along the travelled distance between the slow fluid at the border and the fast fluid at the center seems to contribute to the time course of the solution exchange. This effect would be present in all tubing based devices. Present results might be of fundamental importance for the adequate design of serial compound exchangers which would be instrumental in the discovery of drugs that modulate the action of the physiological agonists of ion channels with the purpose of fine tuning their physiology.

No MeSH data available.


Related in: MedlinePlus