By James Costantin, Ph.D. and Naibo Yang, Ph.D., Molecular Devices Corporation, 3280 Whipple Road, Union City, CA 94587.
The IonWorks Quattro Automated Electrophysiology System can be used in either single-hole (SH) or Population Patch Clamp™ (PPC) modes. We recommend developing assays in SH mode first and then transferring to PPC mode. The IonWorks® System software calculates the arithmetic mean of seal resistances in SH mode as an indicator of the quality of the seals. This calculation is useful in assessing the ability of a population of cells to seal to the (SH) PatchPlate substrate and also allows the user to quickly estimate the sealing reproducibility of a cell line. Since the PPC technique requires that the majority of the 64 holes in each well form adequate seals with cells, it is useful to use the results from a SH experiment to predict the seal resistance expected in a PPC experiment running the same cell line.
The expected PPC well resistance is not, however, the arithmetic mean of the 64 seal resistances in the PPC well. It is the resistance that results from the 64 seal resistances in parallel inside each well. A parallel resistance calculation for 64 individual SH wells can be done in Microsoft Excel® to give an indication of the suitability of a cell line for transfer from SH to PPC mode. The actual parallel resistance for each well in PPC mode is obtained by the inherent analog measurement of the 64 cells in parallel and is displayed in the IonWorks System software as the resistance value for each individual PPC well scaled to represent the resistance per hole.
Plotting the distributions of resistances from SH runs for all 384-wells can also help assess the likelihood of transferring a cell line from a SH to a PPC assay. Figure 1 shows two seal resistance distributions from an IonWorks Quattro System running in SH mode. The single peak distribution shown in Figure 1, Panel A indicates that most of the seals are above 50 MΩ with a uniform distribution centered around 90 MΩ. Cells with this type of seal distribution are a good candidate for transfer from a SH to a PPC assay. The bimodal distribution in Panel B has a similar symmetric distribution with a center around 115 MΩ; however, it also includes a population of cells with very low seals, i.e., < 10 MΩ; these contribute a significant amount of leak conductance and therefore will cause PPC parallel seal resistances to decrease dramatically. We have found it useful to calculate the predicted PPC parallel resistance (Rseal(G)) from SH experimental runs prior to transferring an assay to PPC. A Microsoft Excel template is available from Molecular Devices that calculates six Rseal(G) values for each SH 384-well PatchPlate. The template calculates the mean seal resistance by two different methods; the first is the arithmetic mean (Rseal(R)) using the sum of individual seals/64, and the second is by converting resistance to conductance,
Single-hole (SH) PatchPlate seal distributions are an indicator of suitability for population patch clamp (PPC). A: Cell lines that display a single peak distribution of SH seal resistances are good candidates for transfer from SH to PPC assays. B: Cell lines that display a distribution of single-hole seals with a population of low MΩ values may not transfer well to PPC. The tables give arithmetic mean (Rseal(R)) and parallel values (Rseal(G)) of the single hole seals calculated in groups of 64 and for the whole plate. Rseal(R) and Rseal(G) are calculated as described in the text.
i.e., by calculating the (Rseal(G)) where:
1/Rseal(G) = 1/R1 + 1/R2+ 1/R64
The two histograms in Figure 1 illustrate that a symmetric single peak distribution will give similar results using both methods whereas a bimodal distribution will give very different results for Rseal(R) and Rseal(G).
Before transferring a cell assay from SH to PPC mode, it is useful to examine the SH mode seal distributions in histogram format and to also calculate the expected value of Rseal(G). This can be calculated with the supplementary Microsoft Excel spreadsheet provided by Molecular Devices.
Simulation describing PPC seal resistances
Since a single parallel measurement is made in a PPC well from multiple recording sites and multiple cells, there is no way to determine what the values of the seals are in the individual branches of the parallel circuit. To address this, we have provided two simulations (Figure 2) to help estimate the proportion of “successful” seals, partially occluded holes and completely open holes within the parallel circuit. The first simulation (plotted in brown) assumes that there are only two populations of seals at the recording sites, one being successful seals (120 MΩ) and the other completely open holes (3 MΩ). As the seal rate drops from 100%, the mean seal resistance for the parallel circuit drops off sharply. The measured range in PPC seals that we typically see (50–120 MΩ) corresponds to less than 3% of the holes being completely open (or 2 of 64 holes). The second simulation (plotted in blue) also assumes two populations of seals at the recording sites, one being successful seals (120 MΩ) as in the first simulation, and the other population being partially occluded holes (assumed to be 10 MΩ). In this simulation, the measured range in PPC seals that we typically see corresponds to less than 12% of the holes (7 of 64) being partially occluded. The actual situation probably resembles something in between the two simulations. There is probably a mixture of successful seals, partially occluded holes, and completely open holes. In any case, both simulations suggest that the seal rates must be 88% or greater, which is not difficult to routinely achieve once a cell line is optimized.
Two simulations to estimate the number of successful seals, partially occluded holes, and completely open holes. Prediction of percentage of open holes assuming leak for properly clamped cell is 120 MΩ and all other holes are partially occluded at 10 MΩ or completely open at 3 MΩ.