Downloaded from http://cshprotocols.cshlp.org/ at NYU MED CTR LIBRARY on February 19, 2015 - Published by Cold Spring Harbor Laboratory Press
Protocol
Cell-Flow Technique George P. Hess, Ryan W. Lewis, and Yongli Chen
Various devices have been used to flow neurotransmitter solutions over cells containing receptors (e.g., ligand-gated ion channels) for whole-cell current recordings. With many of the devices, the orientation between the porthole of the flow device and the cell is not maintained absolutely constant. Orientation is critical for reproducibility in kinetic experiments. To be able to change the composition of the flowing solution during an experiment and still maintain a constant orientation, we use the cell-flow device described here. A peristaltic pump, a stainless steel U-tube, two different sizes of peristaltic tubing, and a solenoid valve are required to create a simple solution exchange system that can rapidly apply and remove solutions over the surface of a cell in tens of milliseconds. This system allows one to test multiple conditions on a cell containing the receptor of interest while constantly “washing” the cell with extracellular buffer solution between experimental applications. The use of the solenoid valve allows for the application of solutions to be precisely timed and controlled by a computer during electrophysiological current recording.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.
Reagents
Buffers (extracellular and intracellular) There are no standardized buffer compositions for electrophysiology or even for particular receptors (Walz et al. 2002). Buffer compositions have several roles, however, and, therefore, properties; they must mimic the osmotic composition of the cytosol or extracellular environment, provide the ions needed for studying the receptor of interest and limit the conductance of ion channels that may be present in the cell membrane but that are not being studied. The pH of the solutions should be 7, and the buffering capacity should be adequate if photolysis is likely to cause changes in the value. Deionized, distilled water should be used for all solutions. Buffers should be passed through sterile 0.22-µm filters and stored in sterile containers.
Cultured cells expressing the receptor of interest The cells used for whole-cell current recordings either must express the receptor of interest or be transfected with cDNAs leading to the expression of the receptor of interest. We have used primary cells, the PC12 cell line with sympathetic ganglionic nicotinic acetylcholine receptors, the BC3H1 cell line with endogenous muscle-type nicotinic acetylcholine receptors, and HEK293T cells transiently transfected with cDNAs encoding receptor subunits of interest. In the case of HEK293T cells transiently expressing recombinant receptors, we often cotransfect a plasmid with the cDNA for green fluorescent protein as a transfection marker. The cells are typically seeded and grown for at least 24 h on 35-mm cell culture dishes under the optimal conditions for the particular cells. The health of the cells, indicated, for example, by “normal” cell morphology and a sharp/clearly defined cellular membrane, is critical for successful whole-cell patch-clamping and current recording.
Adapted from Imaging: A Laboratory Manual (ed. Yuste). CSHL Press, Cold Spring Harbor, NY, USA, 2011. © 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot084160
1092
Downloaded from http://cshprotocols.cshlp.org/ at NYU MED CTR LIBRARY on February 19, 2015 - Published by Cold Spring Harbor Laboratory Press
Cell-Flow Technique
Equipment
Analog-to-digital converter (“digitizer”; Molecular Devices, Digidata 1322A) Antivibration, floating table (table or table top) Cell culture dishes (35-mm; Corning) Color-coded peristaltic pump tubing (Elkay, Krackler Scientific, or Cole Parmer) For example, use 0.25-mm inner diameter (orange-blue) tubing for the application of the solution combined with 0.38-mm inner diameter (orange-green) tubing for the suction side (or use tubing with comparable size ratios).
Computer with appropriate sampling/acquisition software A computer (with electrophysiological software for data acquisition, e.g., Clampex), digitizer, and oscilloscope (optional) are connected to a patch-clamp amplifier.
Gilson Minpuls 3 peristaltic pump, or similar Head stage (Molecular Devices) A head stage connected to the amplifier is the connection point for the grounding electrode and the recording electrode/pipette holder (see Fig. 1A). The entire head stage is mounted onto a micromanipulator, allowing for precise moment and positioning of the recording pipette when creating a membrane gigaohm seal.
Inverted microscope setup within a Faraday cage An inverted microscope with good working distance above the stage is set on a vibration-resistant table and is surrounded by a Faraday cage to diminish background electromagnetic noise. A copper cage is ideal, but aluminum window screens can be used to make an inexpensive cage. If possible, place power supplies and lamps outside the Faraday cage to reduce noise and turn them off when not needed during current recording. It is critical that all the equipment is electrically grounded. Electrically grounding the Faraday cage, microscope, and other equipment to a single ground source helps to eliminate ground loops and minimizes background noise.
Microforge (Narishige, MF-830) Micromanipulators (Narishige) Oscilloscope (optional)
A
B 200 μm U-tube 150 μm
5 4 10 1
11
100 μm
Optical fiber
Cell
Recording pipette
25 μm
2 3
12
6 7
9
Direction of flowing solution
Reference electrode
8
FIGURE 1. (A) A schematic drawing of the device used for the cell-flow technique. The components shown include the stainless steel U-tube (1), the fiber optic cable (2; not used for the cell-flow technique, but required for the related flash/ laser-pulse photolysis technique; see Caged Neurotransmitters and Other Caged Compounds: Design and Application [Hess et al. 2014]), a borosilicate recording pipette containing intracellular buffer and the recording electrode (3), the pipette holder (4), the head stage (5), the suction/vacuum tube (6), the reference electrode (7), the microscope (objective) for viewing cells and aligning the U-tube (8), a cell culture plate with cells expressing the receptor of interest (9), a three-port solenoid valve (10), 0.38- or 0.42-mm inner-diameter peristaltic tubing drawing solution away from the U-tube (11), and 0.25- or 0.5-mm inner-diameter peristaltic tubing with solution flowing toward the U-tube (12). (B) A zoomed-in diagram depicting the alignment of components. (Again, the optical fiber is not used for the cell-flow technique.) The porthole has a diameter of 150 µm and is placed 100–200 µm from a cell suspended from the recording electrode. While the solenoid valve is open, solution is actively drawn away from the U-tube at a higher rate than that at which solution flows into the U-tube. The U-tube draws extracellular buffer in through the porthole from the dish, preventing any leakage or diffusion of ligand solution over the cell. When the solenoid valve closes, solution being pumped to the U-tube is forced out of the U-tube porthole and over the surface of the cell. Linear flow rates of 1– 4 cm/sec are typically used. A more rapid flow is a disadvantage because the integrity of the whole-cell seal between the cell and the electrode tends to deteriorate.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot084160
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G.P. Hess et al.
Patch-clamp amplifier (Molecular Devices, Axopatch 200B) Pipette holder The pipette holder is connected to a vacuum tube with a closing valve (see Fig. 1A). Inclusion of a small 25- to 50mL flask in the vacuum tube line increases the control over the degree of vacuum one can draw on the pipette. Vacuum can be applied to the line either by mouth or by a syringe, depending on the preference of the experimenter.
Recording and reference electrodes Recording and reference electrodes are predominately made of silver wire that has been chloride coated by either electroplating or chemical (bleach) treatment (Sakmann and Neher 1995). Both methods seem to work equally well, and the coating helps to stabilize the open electrode potential.
Recording pipettes of suitable dimensions Recording pipettes are made from borosilicate capillaries (World Precision Instruments); the dimensions used are dependent on the currents to be measured. For instance, for GABAA receptors we use capillaries with a 1.5-mm outer diameter and a 1.12-mm internal diameter. Pipettes can be made using a pipette puller (HEKA, PIP5), which can be programmed to pull pipettes of different geometries. We use a two-stage vertical pipette puller, finding this preferable to a horizontal puller, but either will work. The tips of the pulled pipettes are heat-polished (fire-polished) on a microforge. Heat-polishing is not required, but we find that it increases the likelihood of forming a stable gigaohm seal with the cell.
Solenoid valve (Lee valve; Lee Co.) Stainless steel Hamilton syringe needle tubing (outer diameter 300–400 µm; inner diameter 200–300 µm) for creation and assembly of the U-tube (for details, see Step 1) Stainless steel tubing is used because the internal surface is more uniform than that of plastic or glass tubing.
Stand to hold manipulator and U-tube arm U-tube arm with a clamp METHOD Making the U-Tube and Setting Up the Peristaltic Pump
1. Create a U-tube by bending the stainless steel Hamilton syringe needle tubing into a U-shape with a distance between the arms of 5 mm. Drill a porthole with a diameter of 150 µm at the apex of the tube for solution to flowthrough (Fig. 1B). 2. Clamp the U-tube to a U-tube arm made of Plexiglas [poly(methyl 2-methylpropenoate)] to minimize electrical noise. To allow adjustments in the position of the U-tube during testing, insert the arm into a coarse manipulator that fastens either to the microscope or to the vibrationresistant table. 3. Set up the connections (shown in Fig. 1A). i. Connect the inlet of the U-tube to the narrower peristaltic tubing with an internal diameter of 0.25 mm. ii. Connect the outlet of the U-tube by a very short (3- to 6-cm) tubing, also with an internal diameter of 0.25 mm, to the inlet of the solenoid valve. iii. Connect the outlet of the solenoid valve to the wider peristaltic tubing with a large internal diameter of 0.38 mm. This larger diameter causes a larger volume of solution to be drawn from the U-tube than the volume sent to the U-tube, thus causing extracellular buffer to be drawn through the porthole into the U-tube and preventing any experimental solutions from leaking onto the cell.
4. Place both the inlet and outlet peristaltic tubes onto the peristaltic pump head such that the inlet (narrower) and the outlet (wider) tubing flow solution to and from the U-tube, respectively. Using a syringe, fill the tubing with water, clamping the tubes on the peristaltic pump head, and insert the inlet tubing into the solution desired to run through the U-tube and the outlet tubing into a waste container. 1094
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot084160
Downloaded from http://cshprotocols.cshlp.org/ at NYU MED CTR LIBRARY on February 19, 2015 - Published by Cold Spring Harbor Laboratory Press
Cell-Flow Technique
5. Place a 35-mm dish containing water on the microscope stage, submerge the U-tube, and turn on the peristaltic pump. The flow of solutions should be as smooth as possible. Test the flow by allowing a small air bubble into the inlet tubing. The bubble should move at a smooth and constant rate, and the best place to look for this is in the tubing between the peristaltic pump head and the U-tube, where the solution is under slight back pressure. Fluctuations in the flow rate are usually caused by improper tightening of the peristaltic pump clamps. Overtightening not only causes poor and uneven flow of testing solutions out of the U-tube porthole, but it also decreases the lifetime of the peristaltic tubing.
6. Calculate and/or measure the solution flow rate, and adjust the rate to achieve a balance between rapid solution exchange and a gentle flow that will not damage a cell being tested. 7. Connect the solenoid valve with a BNC cable to a digital output on the digitizer. The solenoid valve can be controlled through the use of data acquisition software such as Clampex. Design an episodic protocol in the software that will switch the solenoid valve on for a given number of seconds (e.g., 1–4 sec), causing the water to flow out of the U-tube porthole, and then switch it off again. This step is used to test that the U-tube system is working correctly (as in Steps 8–14) and for the application of experimental solutions to a cell.
Testing the Flow of Solution Now that the U-tube flow system is set up, it is important to test that the solution flowing through the U-tube and out of the porthole exchanges rapidly and is consistent.
8. Turn on the pump and draw distilled, deionized water through the inlet tubing. 9. Submerge the U-tube in a dish of extracellular buffer (replacing the dish of water), and bring the porthole of the U-tube into focus under the microscope. 10. Backfill a recording pipette with intracellular buffer, and insert the filled pipette into the pipette holder on the head stage. 11. Lower the tip of the pipette into the extracellular buffer in the dish, and bring it into focus using the coarse manipulator holding the head stage, making sure the electrode is in the buffer. 12. Using the fine manipulator, center the tip of the open-ended pipette 100–200 µm in front of the U-tube porthole. Release any back pressure within the pipette holder that may be pushing solution out of the pipette tip by opening and then closing the valve used for applying a vacuum. Make sure the reference (grounding) electrode is also in the dish. 13. Set (but do not yet turn on) the holding potential of the amplifier to a moderate voltage, such as −60 mV. While in the voltage-clamp mode and metering current, adjust the resting conductance to 0 on the amplifier. Now switch on the negative holding potential; there should be a large negative conductance. 14. Run the acquisition software protocol created in Step 7 to close the solenoid valve for a short period while recording the current. If the flow is ideal, the current should sharply increase toward 0 pA, followed by a flat conductance level and then a rapid decrease in conductance back to the original conductance level at times corresponding to the protocol. This change in conductance is caused by the flowing of nonconductive, pure deionized water out of the U-tube porthole and over the tip of the recording pipette. Ideally, the rising phase of the conductance change from 10% to 90% should only span 10–20 msec. If the flow is constant and without pulsing or pausing, the current observed should be perfectly flat during the application time.
15. If the flow looks good, start the flow of experimental solutions over a cell. Turn off the pump, and change the solutions to those to be used for measurements. To maintain and clean the U-tube system, it is a good practice to wash the tubing with distilled/deionized water after conducting experiments. This prevents the buildup of salt deposits and the growth of microbes within the tubing and the solenoid valve that may lead to irregularities in the flow of solutions.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot084160
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G.P. Hess et al.
Cell-Flow Measurements
16. Place a 35-mm culture dish of adherent cells in extracellular buffer on the microscope stage. Select a cell, establish a whole-cell patch-clamp configuration with the cell: i. Backfill a borosilicate recording pipette with intracellular buffer and insert it into the pipette holder by sliding it carefully over the recording electrode. ii. Create a gigaohm seal between the pipette and the membrane of a cell by positioning the pipette on the surface of the cell membrane and applying a small amount of vacuum on the inside of the pipette through a tube connected to the pipette holder. iii. Convert the cell-attached state into a whole-cell configuration by breaking the membrane between the cytosol and the intracellular buffer of the pipette with a brief and sharp increase in vacuum or a brief voltage transient. iv. Voltage-clamp the cell with the patch-clamp amplifier to the desired potential across the cell membrane. 17. While the cell is in the whole-cell configuration, gently lift the cell from the surface of the cell culture dish using the micromanipulator. This may require a great deal of patience for some cell types.
18. Lower the U-tube into the extracellular buffer in the dish, and turn on the peristaltic pump to start the flow of ligand solution through the U-tube. 19. Using the microscope and micromanipulators, bring into focus both the U-tube porthole and the cell at the end of the recording pipette. When both of these are in focus, the U-tube and the cell are aligned on the same vertical plane. Center the cell 100–200 µm in front of the porthole of the U-tube. 20. Run a desired protocol in the acquisition software to apply the ligand solution to the cell suspended from the recording pipette. RELATED INFORMATION
For further discussion of this technique, its development, and its uses, see Udgaonkar and Hess (1987) and Caged Neurotransmitters and Other Caged Compounds: Design and Application (Hess et al. 2014). REFERENCES Hess GP, Lewis RW, Chen Y. 2014. Caged neurotransmitters and other caged compounds: Design and application. Cold Spring Harb Protoc doi: 10.1101/pdb.top084152. Sakmann B, Neher E, eds. 1995. Single-channel recording, 2nd ed. Plenum, New York. Udgaonkar JB, Hess GP. 1987. Chemical kinetic measurements of a mammalian acetylcholine receptor by a fast-reaction technique. Proc Natl Acad Sci 84: 8758–8762.
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Walz W, Boulton AA, Baker GB, eds. 2002. Patch-clamp analysis: Advanced techniques. In Neuromethods (series eds. Boulton AA, Baker GB), Vol. 35. Humana Press, Totowa, NJ.
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Downloaded from http://cshprotocols.cshlp.org/ at NYU MED CTR LIBRARY on February 19, 2015 - Published by Cold Spring Harbor Laboratory Press
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Protocol
Cell-Flow Technique George P. Hess, Ryan W. Lewis, and Yongli Chen
Various devices have been used to flow neurotransmitter solutions over cells containing receptors (e.g., ligand-gated ion channels) for whole-cell current recordings. With many of the devices, the orientation between the porthole of the flow device and the cell is not maintained absolutely constant. Orientation is critical for reproducibility in kinetic experiments. To be able to change the composition of the flowing solution during an experiment and still maintain a constant orientation, we use the cell-flow device described here. A peristaltic pump, a stainless steel U-tube, two different sizes of peristaltic tubing, and a solenoid valve are required to create a simple solution exchange system that can rapidly apply and remove solutions over the surface of a cell in tens of milliseconds. This system allows one to test multiple conditions on a cell containing the receptor of interest while constantly “washing” the cell with extracellular buffer solution between experimental applications. The use of the solenoid valve allows for the application of solutions to be precisely timed and controlled by a computer during electrophysiological current recording.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.
Reagents
Buffers (extracellular and intracellular) There are no standardized buffer compositions for electrophysiology or even for particular receptors (Walz et al. 2002). Buffer compositions have several roles, however, and, therefore, properties; they must mimic the osmotic composition of the cytosol or extracellular environment, provide the ions needed for studying the receptor of interest and limit the conductance of ion channels that may be present in the cell membrane but that are not being studied. The pH of the solutions should be 7, and the buffering capacity should be adequate if photolysis is likely to cause changes in the value. Deionized, distilled water should be used for all solutions. Buffers should be passed through sterile 0.22-µm filters and stored in sterile containers.
Cultured cells expressing the receptor of interest The cells used for whole-cell current recordings either must express the receptor of interest or be transfected with cDNAs leading to the expression of the receptor of interest. We have used primary cells, the PC12 cell line with sympathetic ganglionic nicotinic acetylcholine receptors, the BC3H1 cell line with endogenous muscle-type nicotinic acetylcholine receptors, and HEK293T cells transiently transfected with cDNAs encoding receptor subunits of interest. In the case of HEK293T cells transiently expressing recombinant receptors, we often cotransfect a plasmid with the cDNA for green fluorescent protein as a transfection marker. The cells are typically seeded and grown for at least 24 h on 35-mm cell culture dishes under the optimal conditions for the particular cells. The health of the cells, indicated, for example, by “normal” cell morphology and a sharp/clearly defined cellular membrane, is critical for successful whole-cell patch-clamping and current recording.
Adapted from Imaging: A Laboratory Manual (ed. Yuste). CSHL Press, Cold Spring Harbor, NY, USA, 2011. © 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot084160
1092
Downloaded from http://cshprotocols.cshlp.org/ at NYU MED CTR LIBRARY on February 19, 2015 - Published by Cold Spring Harbor Laboratory Press
Cell-Flow Technique
Equipment
Analog-to-digital converter (“digitizer”; Molecular Devices, Digidata 1322A) Antivibration, floating table (table or table top) Cell culture dishes (35-mm; Corning) Color-coded peristaltic pump tubing (Elkay, Krackler Scientific, or Cole Parmer) For example, use 0.25-mm inner diameter (orange-blue) tubing for the application of the solution combined with 0.38-mm inner diameter (orange-green) tubing for the suction side (or use tubing with comparable size ratios).
Computer with appropriate sampling/acquisition software A computer (with electrophysiological software for data acquisition, e.g., Clampex), digitizer, and oscilloscope (optional) are connected to a patch-clamp amplifier.
Gilson Minpuls 3 peristaltic pump, or similar Head stage (Molecular Devices) A head stage connected to the amplifier is the connection point for the grounding electrode and the recording electrode/pipette holder (see Fig. 1A). The entire head stage is mounted onto a micromanipulator, allowing for precise moment and positioning of the recording pipette when creating a membrane gigaohm seal.
Inverted microscope setup within a Faraday cage An inverted microscope with good working distance above the stage is set on a vibration-resistant table and is surrounded by a Faraday cage to diminish background electromagnetic noise. A copper cage is ideal, but aluminum window screens can be used to make an inexpensive cage. If possible, place power supplies and lamps outside the Faraday cage to reduce noise and turn them off when not needed during current recording. It is critical that all the equipment is electrically grounded. Electrically grounding the Faraday cage, microscope, and other equipment to a single ground source helps to eliminate ground loops and minimizes background noise.
Microforge (Narishige, MF-830) Micromanipulators (Narishige) Oscilloscope (optional)
A
B 200 μm U-tube 150 μm
5 4 10 1
11
100 μm
Optical fiber
Cell
Recording pipette
25 μm
2 3
12
6 7
9
Direction of flowing solution
Reference electrode
8
FIGURE 1. (A) A schematic drawing of the device used for the cell-flow technique. The components shown include the stainless steel U-tube (1), the fiber optic cable (2; not used for the cell-flow technique, but required for the related flash/ laser-pulse photolysis technique; see Caged Neurotransmitters and Other Caged Compounds: Design and Application [Hess et al. 2014]), a borosilicate recording pipette containing intracellular buffer and the recording electrode (3), the pipette holder (4), the head stage (5), the suction/vacuum tube (6), the reference electrode (7), the microscope (objective) for viewing cells and aligning the U-tube (8), a cell culture plate with cells expressing the receptor of interest (9), a three-port solenoid valve (10), 0.38- or 0.42-mm inner-diameter peristaltic tubing drawing solution away from the U-tube (11), and 0.25- or 0.5-mm inner-diameter peristaltic tubing with solution flowing toward the U-tube (12). (B) A zoomed-in diagram depicting the alignment of components. (Again, the optical fiber is not used for the cell-flow technique.) The porthole has a diameter of 150 µm and is placed 100–200 µm from a cell suspended from the recording electrode. While the solenoid valve is open, solution is actively drawn away from the U-tube at a higher rate than that at which solution flows into the U-tube. The U-tube draws extracellular buffer in through the porthole from the dish, preventing any leakage or diffusion of ligand solution over the cell. When the solenoid valve closes, solution being pumped to the U-tube is forced out of the U-tube porthole and over the surface of the cell. Linear flow rates of 1– 4 cm/sec are typically used. A more rapid flow is a disadvantage because the integrity of the whole-cell seal between the cell and the electrode tends to deteriorate.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot084160
1093
Downloaded from http://cshprotocols.cshlp.org/ at NYU MED CTR LIBRARY on February 19, 2015 - Published by Cold Spring Harbor Laboratory Press
G.P. Hess et al.
Patch-clamp amplifier (Molecular Devices, Axopatch 200B) Pipette holder The pipette holder is connected to a vacuum tube with a closing valve (see Fig. 1A). Inclusion of a small 25- to 50mL flask in the vacuum tube line increases the control over the degree of vacuum one can draw on the pipette. Vacuum can be applied to the line either by mouth or by a syringe, depending on the preference of the experimenter.
Recording and reference electrodes Recording and reference electrodes are predominately made of silver wire that has been chloride coated by either electroplating or chemical (bleach) treatment (Sakmann and Neher 1995). Both methods seem to work equally well, and the coating helps to stabilize the open electrode potential.
Recording pipettes of suitable dimensions Recording pipettes are made from borosilicate capillaries (World Precision Instruments); the dimensions used are dependent on the currents to be measured. For instance, for GABAA receptors we use capillaries with a 1.5-mm outer diameter and a 1.12-mm internal diameter. Pipettes can be made using a pipette puller (HEKA, PIP5), which can be programmed to pull pipettes of different geometries. We use a two-stage vertical pipette puller, finding this preferable to a horizontal puller, but either will work. The tips of the pulled pipettes are heat-polished (fire-polished) on a microforge. Heat-polishing is not required, but we find that it increases the likelihood of forming a stable gigaohm seal with the cell.
Solenoid valve (Lee valve; Lee Co.) Stainless steel Hamilton syringe needle tubing (outer diameter 300–400 µm; inner diameter 200–300 µm) for creation and assembly of the U-tube (for details, see Step 1) Stainless steel tubing is used because the internal surface is more uniform than that of plastic or glass tubing.
Stand to hold manipulator and U-tube arm U-tube arm with a clamp METHOD Making the U-Tube and Setting Up the Peristaltic Pump
1. Create a U-tube by bending the stainless steel Hamilton syringe needle tubing into a U-shape with a distance between the arms of 5 mm. Drill a porthole with a diameter of 150 µm at the apex of the tube for solution to flowthrough (Fig. 1B). 2. Clamp the U-tube to a U-tube arm made of Plexiglas [poly(methyl 2-methylpropenoate)] to minimize electrical noise. To allow adjustments in the position of the U-tube during testing, insert the arm into a coarse manipulator that fastens either to the microscope or to the vibrationresistant table. 3. Set up the connections (shown in Fig. 1A). i. Connect the inlet of the U-tube to the narrower peristaltic tubing with an internal diameter of 0.25 mm. ii. Connect the outlet of the U-tube by a very short (3- to 6-cm) tubing, also with an internal diameter of 0.25 mm, to the inlet of the solenoid valve. iii. Connect the outlet of the solenoid valve to the wider peristaltic tubing with a large internal diameter of 0.38 mm. This larger diameter causes a larger volume of solution to be drawn from the U-tube than the volume sent to the U-tube, thus causing extracellular buffer to be drawn through the porthole into the U-tube and preventing any experimental solutions from leaking onto the cell.
4. Place both the inlet and outlet peristaltic tubes onto the peristaltic pump head such that the inlet (narrower) and the outlet (wider) tubing flow solution to and from the U-tube, respectively. Using a syringe, fill the tubing with water, clamping the tubes on the peristaltic pump head, and insert the inlet tubing into the solution desired to run through the U-tube and the outlet tubing into a waste container. 1094
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot084160
Downloaded from http://cshprotocols.cshlp.org/ at NYU MED CTR LIBRARY on February 19, 2015 - Published by Cold Spring Harbor Laboratory Press
Cell-Flow Technique
5. Place a 35-mm dish containing water on the microscope stage, submerge the U-tube, and turn on the peristaltic pump. The flow of solutions should be as smooth as possible. Test the flow by allowing a small air bubble into the inlet tubing. The bubble should move at a smooth and constant rate, and the best place to look for this is in the tubing between the peristaltic pump head and the U-tube, where the solution is under slight back pressure. Fluctuations in the flow rate are usually caused by improper tightening of the peristaltic pump clamps. Overtightening not only causes poor and uneven flow of testing solutions out of the U-tube porthole, but it also decreases the lifetime of the peristaltic tubing.
6. Calculate and/or measure the solution flow rate, and adjust the rate to achieve a balance between rapid solution exchange and a gentle flow that will not damage a cell being tested. 7. Connect the solenoid valve with a BNC cable to a digital output on the digitizer. The solenoid valve can be controlled through the use of data acquisition software such as Clampex. Design an episodic protocol in the software that will switch the solenoid valve on for a given number of seconds (e.g., 1–4 sec), causing the water to flow out of the U-tube porthole, and then switch it off again. This step is used to test that the U-tube system is working correctly (as in Steps 8–14) and for the application of experimental solutions to a cell.
Testing the Flow of Solution Now that the U-tube flow system is set up, it is important to test that the solution flowing through the U-tube and out of the porthole exchanges rapidly and is consistent.
8. Turn on the pump and draw distilled, deionized water through the inlet tubing. 9. Submerge the U-tube in a dish of extracellular buffer (replacing the dish of water), and bring the porthole of the U-tube into focus under the microscope. 10. Backfill a recording pipette with intracellular buffer, and insert the filled pipette into the pipette holder on the head stage. 11. Lower the tip of the pipette into the extracellular buffer in the dish, and bring it into focus using the coarse manipulator holding the head stage, making sure the electrode is in the buffer. 12. Using the fine manipulator, center the tip of the open-ended pipette 100–200 µm in front of the U-tube porthole. Release any back pressure within the pipette holder that may be pushing solution out of the pipette tip by opening and then closing the valve used for applying a vacuum. Make sure the reference (grounding) electrode is also in the dish. 13. Set (but do not yet turn on) the holding potential of the amplifier to a moderate voltage, such as −60 mV. While in the voltage-clamp mode and metering current, adjust the resting conductance to 0 on the amplifier. Now switch on the negative holding potential; there should be a large negative conductance. 14. Run the acquisition software protocol created in Step 7 to close the solenoid valve for a short period while recording the current. If the flow is ideal, the current should sharply increase toward 0 pA, followed by a flat conductance level and then a rapid decrease in conductance back to the original conductance level at times corresponding to the protocol. This change in conductance is caused by the flowing of nonconductive, pure deionized water out of the U-tube porthole and over the tip of the recording pipette. Ideally, the rising phase of the conductance change from 10% to 90% should only span 10–20 msec. If the flow is constant and without pulsing or pausing, the current observed should be perfectly flat during the application time.
15. If the flow looks good, start the flow of experimental solutions over a cell. Turn off the pump, and change the solutions to those to be used for measurements. To maintain and clean the U-tube system, it is a good practice to wash the tubing with distilled/deionized water after conducting experiments. This prevents the buildup of salt deposits and the growth of microbes within the tubing and the solenoid valve that may lead to irregularities in the flow of solutions.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot084160
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Cell-Flow Measurements
16. Place a 35-mm culture dish of adherent cells in extracellular buffer on the microscope stage. Select a cell, establish a whole-cell patch-clamp configuration with the cell: i. Backfill a borosilicate recording pipette with intracellular buffer and insert it into the pipette holder by sliding it carefully over the recording electrode. ii. Create a gigaohm seal between the pipette and the membrane of a cell by positioning the pipette on the surface of the cell membrane and applying a small amount of vacuum on the inside of the pipette through a tube connected to the pipette holder. iii. Convert the cell-attached state into a whole-cell configuration by breaking the membrane between the cytosol and the intracellular buffer of the pipette with a brief and sharp increase in vacuum or a brief voltage transient. iv. Voltage-clamp the cell with the patch-clamp amplifier to the desired potential across the cell membrane. 17. While the cell is in the whole-cell configuration, gently lift the cell from the surface of the cell culture dish using the micromanipulator. This may require a great deal of patience for some cell types.
18. Lower the U-tube into the extracellular buffer in the dish, and turn on the peristaltic pump to start the flow of ligand solution through the U-tube. 19. Using the microscope and micromanipulators, bring into focus both the U-tube porthole and the cell at the end of the recording pipette. When both of these are in focus, the U-tube and the cell are aligned on the same vertical plane. Center the cell 100–200 µm in front of the porthole of the U-tube. 20. Run a desired protocol in the acquisition software to apply the ligand solution to the cell suspended from the recording pipette. RELATED INFORMATION
For further discussion of this technique, its development, and its uses, see Udgaonkar and Hess (1987) and Caged Neurotransmitters and Other Caged Compounds: Design and Application (Hess et al. 2014). REFERENCES Hess GP, Lewis RW, Chen Y. 2014. Caged neurotransmitters and other caged compounds: Design and application. Cold Spring Harb Protoc doi: 10.1101/pdb.top084152. Sakmann B, Neher E, eds. 1995. Single-channel recording, 2nd ed. Plenum, New York. Udgaonkar JB, Hess GP. 1987. Chemical kinetic measurements of a mammalian acetylcholine receptor by a fast-reaction technique. Proc Natl Acad Sci 84: 8758–8762.
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Walz W, Boulton AA, Baker GB, eds. 2002. Patch-clamp analysis: Advanced techniques. In Neuromethods (series eds. Boulton AA, Baker GB), Vol. 35. Humana Press, Totowa, NJ.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot084160
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Cell-Flow Technique George P. Hess, Ryan W. Lewis and Yongli Chen Cold Spring Harb Protoc; doi: 10.1101/pdb.prot084160 Email Alerting Service Subject Categories
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