PatchSettings Tutorial

PATCHSETTINGS TUTORIAL

Click one of the below links to view the PatchXpress 7000A PatchSettings Tutorial:


PatchSettings 1: Initialize SealChip

PatchSettings 2: Add Cells

PatchSettings 3: Detect Cell

PatchSettings 4: Sealing

PatchSettings 5: Whole-Cell

PatchSettings 6: Improve Whole Cell

PatchSettings 7: In Procedure

PatchSettings 1: Initialize SealChip

PatchSettings 1: Initialize SealChip

In spite of there being only one setting in this dialog, there are a number of important things to point out here. First is that crosstalk checking is done before the extracellular chamber is filled with solution. If you receive a message that open-circuit resistance was low, or crosstalk was found at this point, then the electrode plate should be inspected for signs of fluid. Spills may occur when the electrode plate is sponged off. If too much fluid is blown into the sponge, or if there is a droplet of solution left over on either gasket surface during the filling, the electrode plate as well as the gasket must be cleaned. Be sure to ground yourself to discharge any static before touching the electrode plate.


The chambers are first washed for 15 seconds prior to reading their Re values. This ensures that all bubbles are removed from the chamber and from the segment of tubing between the reference electrodes and the SealChip chamber. The reference electrodes are located under the metal number plate on the wash station.


The electrodes are nulled twice. Once as soon as the chambers are filled, and once after answering the “add cells now” dialog. Occasionally take a look in your Params files (from DataXpress, right click on the “procedure” under the procedures tab and View-in-Clampfit) for these two tag items to find out how much electrode drift that channel has. Electrode drift may result in offset reversal potentials by the end of a run, and may produce artefactual data. The PatchXpress manual contains a maintenance schedule that provides information on when and how to change the electrode plate.


There are two tests executed at this The first is for low Re chambers. The default cut-off for low Re is 1.0 MO, which corresponds to the limits imposed by the headstage’s feedback resistor (maximum range is -20 to +20 nA). The low Re limit serves to protect the pressure controllers from leaky or broken chambers, otherwise the cell-attraction pressure may draw buffer into the pressure controllers. The high Re setting is programmable in the “Test” section. This setting should always be set to a value at least 20% lower than the resistance that must be achieved to detect whether a cell has landed on the aperture, in tab 3’s Test section, and at least 100% higher than the maximum resistance expected for an acceptable electrode (value will vary with solution contents).


PatchSettings 2: Add Cells

PatchSettings 2: Add Cells

2.1 Apply cell dispersion pressure. This pressure value should always be a positive value, and serves two functions. First it ensures that the IS solution does not get contaminated by ES. The osmotic differences (optimally ~5 mOsm lower in IS) between the solutions are not sufficient to counter the combined effects of gravity (negligible) and capillary action (pulling solution into the electrode tube). In practice, the AVIVA/Axon IS and PBS combination requires at ~+2 mmHg to be applied to result in no net flow through the SealChip aperture. Second, the positive pressure prevents unintended landing of debris in the aperture. The IS solution stored within the tubing is primed with each SealChip, however ES solution must be primed via the SealChip chamber. Any particles that may accumulate within the tubing would otherwise be drawn into the recording aperture and prevent interaction with the cell membranes. Further, during cell delivery, the scraping action resulting from the pipette tip deflecting on the side of the chamber can produce cellular debris that will be equidistant to the recording aperture once cells are delivered. The ejection of cells may itself cause premature contact with the recording aperture if there is not outflow from the aperture. Values from +5 to +10 mmHg have produced the best results.


2.2 Pause to request fresh cells . After the SealChip is prepared and offset potential has been nulled, cell-addition is added to the queue. This selection box inserts a pause and prompt for information line before the cell-addition step is queued. After selecting OK to this prompt, the chamber is washed once more to remove any osmotic effects of evaporation, and electrode offset potentials will be nulled once again. Occasionally take a look in your Params files (from DataXpress, right click on the “procedure” under the procedures tab and View-in-Clampfit) for the two electrode offset value tag items to find out how much electrode drift each channel has.


2.6 Triturate cells. With 3 µL per chamber delivery volume (setting 2.8), the usual volume used for trituration is 3 x 18 + 5 = 59 µL (enough for 18 chambers, with an extra 5 µL) with an extra 5 µL drawn that will be left over after delivery as dead volume. The pipette tip should already be adjusted to be 1mm above the bottom of the eppendorff tube, therefore an extra 10 to 20 µL is necessary to ensure bubbles are not drawn during trituration, for a total required cell suspension volume of at least 85 µL. The number of repeats of the trituration should be increased if the cell suspension total volume will be much greater than twice the minimum requirement (i.e., use 5x trituration if using 200 µL of cell suspension, or more if using a slow trituration rate). The rate should be fast, but limited by the robustness of the cell membrane. For wild-type RBL cells, a rate of 80 to 100 µL/sec has produced the best results.


2.7 There are three timers used throughout an experiment: 1) Experiment time is the time since clicking on the RUN button to start an experiment; 2) SealChip time is the time since picking up a SealChip (it is possible to use multiple SealChips per experiment); 3) Cells added time is the time since the first chamber received cells.



2.8 Add cells to SealChip chambers. The volume that may be added is limited by the size of the pipette tips. The optimal number of cells that should be added to each chamber has been studied at both AVIVA and Axon. AVIVA has found an optimum at 10,000 to 20,000 cells per chamber, and Axon has reported recently that as low as 2,000 cells per chamber is as successful as 20,000 cells per chamber. An ideal number may therefore be 5,000 to 10,000 cells per chamber. The rate of delivery should be as low as may be reliably added with the injector pump, however fast enough that cells within the pipette tip are not allowed to settle by gravity before reaching the 16th chamber. A delivery rate of 3 or 4 µL/sec has been used with good success. Yuri Osipchuk (Axon wet lab) has videotaped the time required for cells to reach the vicinity of the recording aperture during delivery, and found that a mere 100 to 200 msec are sufficient, therefore faster delivery rates are unnecessary, and may additionally create debris by rupturing or damaging some cells during delivery.


2.9 Wait at cell dispersion pressure. This value works in conjunction with settings 3.1 and 3.2 of the next tab. Immediately after cell delivery is complete, the pressure will remain at the value set in setting 2.1 for this amount of time before starting the pressure protocol for cell landing. Since the cells arrive at the vicinity of the recording aperture almost instantly during delivery, this value should be kept very short. Smaller particles, fragments, or damaged cells tend to have a larger surface area to volume ratio and hence are more affected by drag forces in a flowing medium. This duration serves to clear small cellular fragments and contaminating particles from the aperture before the landing pressure is applied.


2.10 Maximum cell settling time. This value uses the Cells-added timer. The value should allow sufficient time for all the cells to land, but not be so long as to inhibit the first procedure from starting. A typical algorithm is to sum the values of Setting(2.9) + Setting(3.2) + Setting(3.4.2) + Setting(3.5-sec) + Setting(5.2.3) + Setting(6.4), then add an extra 5 seconds to ensure time for landing.


PatchSettings 3: Detect Cell

PatchSettings 3: Detect Cell

3.1 Apply cell settling pressure ; and


3.2 Wait for cells to settle fo r. This pressure and time allows for special treatment of the cells to aid in proper cell selection. In cases where the aliquot of cells has a lower cell density, it may be necessary to wait for a short time (1-2 seconds) at low pressure (0-2 torr) to allow the cells to disperse into the vicinity of the hole, without being blown away by the cell dispersion pressure (from setting 2.1). Additionally, such an approach can provide for less contamination of intracellular and extracellular solutions. Alternatively, if higher cell densities are used, this setting may be set to higher pressure (>10 torr) for a short time (3-5 sec) to allow cells that are clustered to settle elsewhere and stick to the surface, while simultaneously reducing the cell load in the vicinity of the hole, thereby reducing the severity of cell layers over the cell that will be the object of the experiment. This latter approach may be titrated to provide cell selection capabilities by taking advantage of the increased drag effect on cells with larger surface-area-to-volume ratios (i.e., smaller cells are more easily blown higher in the chamber than larger cells, therefore a longer settling time under positive pressure should select for smaller cells).


3.3 Apply cell attraction pressure . This value should always be a negative pressure to draw a cell to the aperture and initiate the interaction between the cell and the surface. Ideal values range from -30 to -80 torr and should not be more positive than the value assigned as the peak pressure during the sealing process (in section 4). Higher values promote premature whole-cell access, whereas lower values prolong the sealing time.


3.4 Wait for cell detection. Note that if a cell does not land on the aperture within 90 seconds in a particular chamber, that chamber is switched back to positive pressure and then disabled so as to prevent the negative pressure from drawing the solutions into the pressure controllers. The detection of a cell landing is accomplished by detecting an increase in resistance at the recording aperture that surpasses the test value in this dialog. The test value, for the resistance that the aperture must achieve to determine that a landing has occurred, must be a value greater than the test value in Tab 1, for the resistance that determines whether a chamber is useable. Since there is a finite possibility that the resistance could transiently exceed the test value used to determine a landing without an actual landing having occurred, setting 3.4.2 imposes that the resistance value must be greater than the test value for a minimum amount of time before determining that a cell has landed. Additionally, setting 3.4.2 allows the user to impose a delay before the negative landing pressure is removed. Such a delay may be useful for cells with robust membranes that are more difficult to seal.


3.5 Apply stabilization pressure, holding potential, and wait. Once a landing has been detected, the pressure may be returned to near zero and the cell membrane is allowed to relax for the configured amount of time and press itself against the inside wall of the aperture to promote seal formation.



PatchSettings 4: Sealing

PatchSettings 4: Sealing

Note that in this section it is possible to produce a false gigaseal by applying a -80mV holding potential (membrane test oscillating between -70mV and -90 mV), and loosing the cell such that seal resistance drops below 3.5 MO and saturates the amplifier (-70mV / 3.5 MO = -20nA, but -90mV also produces -20nA because that’s the maximum the amplifier can produce with the 500MO feedback resistor).


4.1 Apply repeating pressure ramps while following rules 4.1.1 and 4.1.2 . The pressure ramps are defined as a peak pressure, ramp duration, hold duration, and time between pulses. The peak pressure should be smaller than the landing pressure (setting 3.3). Part of the function of the landing pressure is to ensure sufficient membrane is drawn into the aperture before it starts to seal. After the landing, however, the pressure should be lowered so as to prevent premature whole-cell access. This is particularly the case in cells that have easy to rupture membranes, such as HEK cells, or when cells are overdigested by enzyme treatment.


The ramp duration, hold duration, and interpulse time should be optimized to each cell type as we have not yet found a reliable set of rules to follow. A good starting point is to keep the ramp and hold times short for cells that are robust but difficult to seal (2 sec and 1 sec, respectively) or for cells that are easy to rupture (4-5 sec and 0 sec, respectively), or longer ramp and hold times for cells that are easy to seal and do not rupture prematurely as easily (10-20 sec and 0-5 sec, respectively). A good starting point for the interpulse time is 10-20 sec for easy to rupture cells that seal easily (e.g., HEK, or overdigested cells), or 5 sec for more robust or difficult to seal cells (e.g., LtK or Jurkat cells).


4.1.1 Skip pressure ramp if seal resistance drops more than : Since not all cells are the same within a population, it is difficult to provide the ideal pressure ramp settings for every cell. For some cells, applying negative pressure will cause a decrease in seal resistance. This setting terminates the pressure ramp when seal resistance drops by more than the set percentage. Although use of a percentage value would seem risky because of what may happen with low seal resistances, with SealChips most landings will produce a seal value of at least 50-200 MO, even when a dead cell or other unwanted particle lands on the aperture, therefore it should be quite safe to set a value here as low as 5-10%. Setting a lower value for this setting will prolong the time to seal for difficult cells, however it will help prevent lost cells and some premature access. Cells with weak membranes that have a propensity for premature access should use lower values (15-20%), whereas more robust cells may use higher values (50%).


4.1.2 Modify holding potential as seal resistance changes . It is commonly known that a negative holding potential will speed the sealing process, however sudden large changes in holding potential may also produce premature whole-cell access. This setting allows for a slow decrease in membrane potential by linking it to the increasing seal resistance.


4.2 Wait until seal resistance threshold is exceeded . This test uses the test values entered under “seal resistance must exceed” and “not drop below”. If cell quality is good, then most holes should achieve 200 MO seals within 5 to 10 seconds, and the majority of holes that seal will do so within 30 seconds. A few cells, however, may take up to 5 minutes to achieve a gigaohm seal, and yet others will get close, but will not achieve a gigaohm seal until after the membrane patch has ruptured to allow whole-cell access. For these cells there is an option for “second chance seal”. Note that the membrane potential will only change between the starting potential from setting 3.5, and the final holding potential from setting 4.6. During sealing, the holding potential will stop following resistance changes once it has reached the final holding level.


4.3 Second chance seal. It may be possible to use cells that did not achieve a gigaohm seal if their whole-cell clamp parameters will still be within acceptable levels to start a procedure. If this option is used, the wait time should be at least 2 minutes to allow the majority of the cells to attain gigaohm seals. After this timeout, whole-cell access may be forced if the new, lower Rseal test threshold value is exceeded. This lower Rseal value should be set to 10 to 20% lower than the minimum acceptable Rm value required to start a procedure (from Tab 6, if used), or it should itself be the minimum Rm value required to start a procedure if the value in Tab 6 is not enabled.


4.4 If a premature whole-cell access is achieved, the membrane potential is immediately set to the final holding potential (in setting 4.6), and the process is moved to step 5.2 as if a normal whole-cell access had been achieved, skipping the remaining pressure ramps.


4.5 Once a seal is detected, the pressure is returned to the stabilization pressure in step 4.3 (note that the value of the pressure is set in 3.5), then monitored for the verification time in 4.4 before reporting a stable seal. This stabilization period is intended merely to prevent false detection of a seal, therefore this duration should be short (1 to 3 seconds). This stabilization period may also be used to ensure a proper seal in cells where the seal resistance decreases significantly when pressure is released (a condition that may also indicate a problem with the voltage clamp solutions, such as pH or osmolarity).


4.6 . This is the location to set the values for stabilizing the seal before achieving whole-cell access. Although most cells may immediately be ruptured to whole-cell access without any loss of seal, some cells must be allowed to stabilize at a slightly negative holding pressure (-3 to -5 mmHg) for 10 to 20 seconds after sealing so that the Rm is not lost immediately after gaining whole-cell access.


PatchSettings 5: Whole Cell

PatchSettings 5: Whole Cell

5.1 Apply Repeating Ramps. The pressure ramps are defined on the right as a starting pressure, a peak pressure, a ramp duration, and a peak hold duration. The goal of this ramp is to gain whole-cell access, therefore the starting pressure should be somewhere near the maximum pressure used for sealing (in Tab 4). This pressure should not yet provide whole-cell access, but should be close, thereby reducing the time to achieve access. The peak pressure may be the maximum vacuum pressure that the pressure controllers can achieve, specifically -300 mmHg. There are, however, good reasons for reducing this peak pressure value, namely when a cell membrane is weak and shows a tendency toward losing membrane resistance upon breaking in. In some cases, higher vacuum pressures can cause the rupture to progress beyond the patch bound by the rim of the aperture, causing leak or loss of access. In other cases, the higher vacuum pressures may draw cell organelles into the aperture and cause an increase in access resistance. We have found that blood cells (Jurkat, RBL) and CHO cells can withstand high break-in pressures without loss or increase in access resistance. HEK cells are more difficult to isolate and often have weaker membranes after isolation, therefore it is safer for these cells to use less than -200 mmHg as the peak pressure.


If a cell patch does not rupture during the first ramp, the ramp will be applied again repeatedly until the timeout listed in tab 6. For this case, it is a good idea to allow at least 10 seconds of pause at the baseline pressure between the ramps. Additionally, there is the option of injecting a quick (0.2 to 0.5 ms) pulse of high energy (1 to 1.5 V) to the patch to facilitate a rupture (“Zap”). Zap before the first pressure ramp should only be used in conditions where the cytoskeleton will not hold the cell contents if break-in ocurrs with vacuum, and zap during the peak pressure should only be used if the cells are particularly difficult to access.


5.2 When Test Conditions Are Met. This step uses the test thresholds for Cm, Ra, and Rm to determine whether whole-cell access has been achieved. The Cm value should usually be set as low as possible to ensure that a transition to access is quickly detected, even if a very small cell has sealed. Selection of cell size, or correction of re-sealed cells should be left to the Ra Optimization and Ra Control protocols. The Cm threshold value should only be increased when working with cells that are known to be homogenous and very large in size, or when the electrode Ce calibrations are incorrectly reporting large Ce values for some chambers. If the latter is the case, it is preferable to correct the Ce values (within the Hardware and Fluidics dialog) rather than to compensate for them in the PatchSettings.


The Ra threshold should only be that which may correctly determine that whole-cell access has been achieved (around 40 to 50 MO for a 15 pF cell). Once again, cell selection by access resistance should be left to Tab 6, or to the procedure itself.


The Rm threshold is hard-programmed into the software at 100 MO. This threshold should suffice for most cell types, however it is important to utilize a holding potential that inactivates or minimizes whole-cell currents upon break-in to ensure that the 100 MO threshold will be triggered.


5.2.1 Hold at Break-in Pressure. The moment access is detected in 5.2, the pressure ramp is cancelled and a verification period is started. Removing the vacuum pressure immediately upon break-in is not always preferable since in many cells there is a good likelihood that the membrane will re-seal. This setting identifies the duration for which the break-in pressure (approximated in software) should be held after access is detected, and before the verification period is started.


Note that the accuracy of the break-in pressure depends on the speed of the ramping. Shorter, faster ramps will produce less accurate approximation of the break-in pressures. While this is irrelevant to this step in the patchsettings, it must be kept in mind when later analyzing your past runs to optimize your PatchSettings values. For cells that are prone to high access resistance values, or to loss of seal upon access, a histogram of the break-in pressures from past experiments should identify the correct value to use as a peak-pressure for the ramps so that better than 80% of the cells will rupture during the first ramp, and the rest will rupture during subsequent ramps. It will also help identify a proper starting-pressure value so that less than 10% of the cells may rupture upon jumping to the starting pressure of the ramps.


5.2.2 Set Procedure Holding Pressure. This value is the holding pressure that will be set during all subsequent optimization and Ra control steps, and will be set at the start of the procedure. This value should normally be set to 0 mmHg, or to between +2 and -5 mmHg to maintain a seal without quickly increasing Ra. Negative pressures between -5 and -30 mmHg have a tendency to increase Ra more quickly, and greater vacuum pressures should only be used if a “corking” action is desired (if, for example, the cell type will never achieve seals greater than 200 MO).


5.2.3 Wait Until Test Verified. This verification period ensures that the cell will not immediately re-seal after gaining access. Although this verification should not take longer than 1 to 2 seconds, this step serves a dual purpose in that it also behaves as a waiting period before the first step of Tab 6, which may potentially apply a strong pressure step. In this case, it is preferable to allow a 5 second pause (or longer) before applying the Ra Optimization protocol in step 6.2.




PatchSettings 6: Improve Whole Cell

PatchSettings 6: Improve Whole Cell


6.1 Add Liquid Junction Potential . As soon as a whole-cell access is confirmed, the holding potential becomes offset by the liquid junction potential (LJP) value that is entered for the pairing of intracellular and extracellular solution that is in use. The value of the LJP is entered within the Solutions dialog (within the Define Experiment dialog):
and within the top section of the dialog, appropriately labelled:

The value of LJP may be measured directly under the conventional electrophysiology technique, or it may be calculated with the help of the LJP calculator supplied within Clampex software that is installed on the PatchXpress computer.


Note for those that attempt to confirm the Vm output from the digitizer that the LJP correction, as well as the electrode offset potential are both added to the analog output stream at the digitizer during the procedure.


6.2 Apply Pneumatic Ra Optimization . The settings for the Ra optimization routine are on the right panel. Upon rupture to whole-cell access there is often a high probability that the membrane may re-seal. A delay is therefore added in tab 5 of the PatchSettings to hold negative pressure for moments longer to avoid resealing. Likewise, access resistance is often not optimal immediately after rupture, however holding the vacuum pressure after rupture often does not reduce Ra as well as pulsing the vacuum pressure. Further, holding a strong negative pressure immediately after break-in may destabilize the seal, and prolonged vacuum pressures may also increase the rate of rise of Ra throughout the experiment. The approach in this optimization routine is to allow the seal to stabilize at low pressure (setting 5.2.3), then to apply short (1 to 3 seconds) negative pressure pulses to the break-in pressure (or to a minimum pressure that is sufficiently strong) to clear the aperture of the chip and ensure low starting Ra value. A quick series of 3 to 4 vacuum pulses repeated every 3 to 5 seconds will usually clear away cellular organelles and membranes or other particles that cause high Ra values. It is important to get the Ra value quickly lowered to its optimum value at the start of the experiment, for if the Ra value is allowed to remain at a high value, it will be more difficult to keep it low during the experiment.


In the case where Rm may be too low (i.e., Rm lost upon gaining whole-cell access), applying further vacuum pulses may force the loss of the seal. If, on the other hand, the seal is not lost, it will often increase over time throughout the experiment and may be useable for screening. To prevent loss of seals, there is the option to define a threshold for Rm or for drop in Rm that will abort the Ra optimization step. The Rm value should be larger (250 to 350 MO) than the minimum acceptable Rm value to start a procedure, and the percentage allowable drop (30 to 50%) should be set to also ensure that Rm does not drop below that acceptable to start a procedure.


6.3 Apply pneumatic Ra Control . There are three options at this point. 1) This option may be disabled. In this case the procedure will start immediately after step 6.4, without any option to hold back the procedure until seal parameters improve. 2) This option may be enabled, with a maximum amount of time in which to attempt to improve the seal parameters before continuing to 6.4. In this case as well the procedure will execute regardless of whether seal parameters are acceptable. 3) This option may be enabled, but told to not allow step 6.4 to execute, and ultimately not to start a procedure until seal parameters are acceptable as defined in the test conditions. Note that PX will keep trying until the timeout for patching process is met. The test settings for Rm and Ra thresholds are the determinants of whether a cell with whole-cell access will be allowed to start an experiment procedure.


6.4 Wait for Stabilization . This value should allow sufficient time to ensure stability of the membrane seal before starting a procedure.


PatchSettings 7: In Procedure


PatchSettings 7: In Procedure

The settings within this tab provide the configurations for two conditions within the procedure. The first is the configuration for whether to use Pneumatic Ra Control within the procedure, and the second is the configuration of whether to terminate cells based on the defined patch health thresholds. These settings are shown in the figure.




Cell Health


Since the Pneumatic Ra Control settings depend on the desired cell health criteria, these will be described first. There are two settings for cell health thresholds. The first defines the minimum acceptable Rm value and the second defines the maximum Ra value. Sealchips are designed to have a starting Rm above 1 GO, and starting Ra below 10 MO.


It is important to note that within the procedure there are the facilities to reassign the cell health thresholds so that one may use a different set of criteria before or after expending the compound. These settings should therefore be as restrictive as possible so as to terminate cells that may not survive long enough to record useable data before any compound is committed. Once compound has been dispensed to the cell, these thresholds should be relaxed within the procedure to ensure that the consumed compound is not wasted.




Minimum Acceptable Rm:


Optimally, the majority of the measured current should represent the current of interest that is the object of the measurements. If, for example, the measurements are to be made at -50 mV, and a typical current is 500 pA, the membrane resistance due to ion channel activity is 100 MO. In this case a leak current of 200 MO would reduce the measured membrane resistance to 66 MO and give an apparent current of 750 pA (+50% error). To achieve < 10% error in this case, it would be necessary to achieve at least 900 MO seal resistance.


Fortunately with many channel types it is possible to either find a potential where the channels are closed, or to adjust the solutions so as to shift the reversal potential such that it is possible to assess the amount of contaminating leak current. In this situation, it should be possible to reduce the Rm threshold back to the 50% error level (200 MO) and apply a post-acquisition correction. If a voltage range is available where the currents are not active, then it is further possible to apply leak correction automatically during acquisition (defined within the procedure). This situation provides the greatest number of useable data, particularly when whole-cell access must be maintained for more than 30 minutes. Where these approaches may not be possible, then although there is no way to compensate for the leak currents, however if the procedure is very short in total duration and if it is reasonable to assume that leak does not change much within the procedure, it may still be possible to use a complete blocker at the end of a procedure to provide the leak subtraction current that can be used post-acquisition. In our hands, when whole-cell access is lost, it drops from ~700 MO quickly down to ~250 MO, then continues to drop more slowly down to ~100 MO or can even stabilize between 150 and 250 MO. An initial value in this setting of >250 MO will usually ensure that those cells that make it into the procedure will last sufficiently long to provide useable data.




Maximum Acceptable Ra:


The ability to clamp membrane potential depends on the ability to drive enough electrons through the aperture per unit of time. The Ra value represents the amount of voltage escape. In the example of a 500 pA current at -50 mV, the amount of voltage escape with 10 MO Ra is only 0.5 mV, therefore the current is well clamped. The threshold for Ra may therefore be set to a higher value to prevent discarding useful data as Ra increases throughout an experiment. The limit should be set by how much voltage change would be required to produce a significant change in membrane current. In the case of a sodium channel, where the current is closer to 1 nA, and where a 2 mV change is sufficient to elicit a significant change in current, the threshold should be set to 2mV / 1nA = 2 MO Ra. In the case of hERG current, where the current is 0.5 nA and a 5 mV change produces significant change in current amplitude, a value of 10 MO Ra should be used.


Note that for the case of sodium channels the desired Ra threshold is approximately equal to the usual electrode resistance (Re) of a Sealchip electrode. Ra is made up of the sum of Re, space-clamp resistance, and steric resistors. It is therefore unreasonable to expect a threshold of 2 MO Ra to be achieved often enough to make experiments feasible. In this case, it is necessary to accept a higher Ra threshold value (as a rule of thumb, Ra is usually ~3x Re), and turn on the electrical Ra correction feature during the procedure.


Pneumatic Ra Control


There are at least two common methods for controlling Ra. One is to apply a negative pressure, then ramp it slowly up or down in response to the changing slope of Ra change, and attenuated to prevent lost seals when Rm is low. The second is to apply short, strong negative pressure pulses, adapted in amplitude to the amount of Ra drop that is needed, but attenuated to prevent lost seals when Rm is low. The PatchXpress software uses the latter method.


This method uses two cell health conditions, one for when the cell is healthy and should be resilient, and one for when the cell is weak and likely easy to loose. These two conditions are defined by the Rm value. The cell is considered healthy and resilient when Rm is above the Rm limit in the High Rm column (in this example it is set to 850 MO), and it is considered poor or weak when Rm is below the Rm limit in the Low Rm column (in this example, 350 MO). Each of the pressure pulse settings that are defined for these two conditions are conserved for all Rm values beyond the respective limits, or vary linearly for all Rm values that fall between these two limits.


In our hands, we have found that cells with Rm below 500 MO are less able to withstand strong pulses of vacuum than those with Rm above 700 MO. The settings of 350 to 850 MO bracket our definitions of good versus poor health so that the ramping pressure pulse settings provide an extra measure of safety by setting safer levels when Rm is much lower, or more aggressive levels when Rm is much higher.


High Rm:


For the High Rm condition, the primary goal of the pneumatic Ra control protocol is to lower Ra when it surpasses a minimum threshold value. That minimum threshold for Ra is defined in the right panel as the target Ra. When Ra is below this value, the pressure control protocol will not be used. Once this target value is surpassed (>8 MO in the example here), pressure pulses of the High Rm duration (2 seconds in the example here) will be applied every High Rm start-to-start interval (10 seconds), at a High Rm minimum pressure amplitude of -40 mmHg. (Note that “Ra low” in the field descriptor refers to the “Target Ra” value.) If Ra continues to increase, however, the pressure pulse amplitude will rise linearly up to the maximum pressure, which is achieved at the Top Ra value (-150 mmHg). (Note that “Ra high” in the field descriptor refers to the “Top Ra” value.)


The goal in this column is to set the Rm value to where you feel confident that the cell can withstand a short but strong pressure pulse that is intended to lower the Ra value by clearing any steric resistors (such as cell organelles) that may be plugging up the access aperture. The two pressure value settings ensure that when only moderate amount of Ra correction is necessary, only moderate pressure is applied, hence preventing the more extreme pressures from being applied needlessly. The higher value is only applied if Ra continues to increase in spite of the pressure protocol.


Note that in our hands pressures between -5 mmHg and -40 mmHg usually result in further increase in Ra value rather than a decrease. This may be because a minimum amount of pressure is necessary to sheer away any steric factors that may be plugging the access.


Low Rm:


For the Low Rm condition, the primary goal of the pneumatic Ra control protocol should be to maintain Rm while trying to reduce Ra. To this end, the pressure values are more moderate (-40 to -60 mmHg), and the pulses are designed to produce less frequent pressure changes (10 second pulses applied every 20 seconds). In our hands, long low pressure pulses are capable of reducing Ra value while maintaining Rm values within our cell health criteria, however this method is less desirable since often the Ra values will climb faster after turning off the pneumatic Ra control protocol. This slower, lower pressure protocol is therefore reserved for when it is most necessary, namely for when the cell Rm is easy to loose.


Cell Specific Changes


The best indicator for optimizing the pneumatic Ra control protocol will come from the data. A history plot of the break-in pressures, ordered from smallest to largest, will reveal a plateau that represents the highest “Minimum pressure” that should be used for the High Rm condition. The lowest value for this same setting should be the lower break-in pressure values that were not from premature accesses. The pulse duration should be kept short, but for some more resilient cells (such as Ltk) the pulses may be 5 or 10 seconds long to achieve best drop in Ra. Weaker cells (such as HEK) should use short (1 sec) pulses, applied less frequently to prevent loss of Rm. Finally, the optimal start-to-start interval should be determined by direct data observation to determine the minimum time required for the membrane Rm and Ra values to stabilize after the pulse, and just start deflecting in one direction or another. The Low Rm settings optima should likewise be extracted from the data, but this time focusing more on cells that had premature whole-cell access.