There is no F in APC: Using physiological fluoride-free solutions for high throughput automated patch clamp experiments

Figures

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  FIGURE 1. HEK and CHO cells recorded in standard and fluoride-free internal solution. (A) HEK cells expressing hERG were captured with almost 80% success rate for RSeal ≥ 1 GΩ + ITail > 150 pA and almost 40% success rate for RSeal ≥ 1 GΩ in fluoride-free internal solution (left). If a QC cutoff of RSeal ≥ 0.25 GΩ + ITail > 150 pA was used, the success rate was increased in both conditions (right). (B) CHO cells expressing NaV1.5 were captured with almost 80% success rate for RSeal ≥ 1 GΩ + IPeak < –100 pA and over 40% success rate for RSeal ≥ 1 GΩ in fluoride-free internal solution (left). If a QC cutoff of RSeal ≥ 0.25 GΩ + IPeak < –100 pA was used, the success rate was increased in both conditions (right). (C) Histogram plot of the RSeal values for HEK-hERG cells in standard and fluoride-free internal solution. For RSeal > 4 GΩ, although they are high giga-seals, the seal resistance is not accurately calculated and is therefore shown as an extra bar and these RSeal values were not included in the calculation for RSeal (Table 2). (D) Histogram plot of the RSeal values for CHO-NaV1.5 cells in standard and fluoride-free internal. For RSeal > 4 GΩ, although they are high giga-seals, the seal…

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  FIGURE 2. hERG currents in standard internal and fluoride-free internal solution. (A) Histogram plot of IPeak values for standard internal and fluoride-free internal. There was a tendency to higher peak amplitudes in fluoride-free internal solution vs. standard internal. (B) Current-voltage traces of hERG using standard internal (left) and fluoride-free internal (right). The voltage protocol is shown underneath. (C) IV curves for an average of 146 cells in standard internal and 319 cells in fluoride-free solution are shown overlaid. A standard Boltzmann equation (see Section “Data analysis”) was used to fit the curve from –120 to –20 mV. (D) Vhalf values for hERG recorded in standard internal and fluoride-free internal. The Vhalf values of 190 cells in standard internal and 261 cells in fluoride-free internal were not significantly different using a Student’s t-test (P > 0.05).

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  FIGURE 3. Pharmacology of hERG recorded in standard and fluoride-free internal solution. (A,B) Raw current traces from an example HEK cell expressing hERG recorded in standard internal (A) and fluoride-free internal (B) solution was blocked by increasing concentrations of verapamil [voltage protocol is shown at the top of panel (A)]. (C) Corresponding time course of block by verapamil in standard internal (C) and fluoride-free internal (D), 54 or 11 cells, respectively. Time course of 58/12 cells in DMSO control is also shown within panels (C,D). (E) Concentration response curve for verapamil in standard internal solution or fluoride-free solution are shown overlaid. (F) IC50s for terfenadine and verapamil recorded using standard internal or fluoride-free internal solution are shown as a box plot. The IC50s for verapamil and terfenadine recorded in standard internal or fluoride-free internal solution were not significantly different using a Student’s t-test (P > 0.05).

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  FIGURE 4. NaV1.5 recorded in standard and fluoride-free internal solution. (A) Histogram plot of IPeak values recorded using standard internal and fluoride-free internal solution. (B) Current-voltage traces of NaV1.5 expressed in CHO cells using an activation protocol (top) and inactivation protocol (bottom) for both conditions. (C) Activation (fit with an extended Boltzmann equation, see Section “Data analysis”) and inactivation IV curves (fit with a Boltzmann equation, see Section “Data analysis”) for NaV1.5 in standard internal and fluoride-free solution are shown overlaid. (D) The Vhalf values of activation of 484 cells in standard internal were significantly more negative compared with 241 cells in fluoride-free internal. The same shift to more negative Vhalf of inactivation was also found for 728 in standard internal fluoride compared with 165 cells in fluoride-free internal (Student’s t-test, ***P < 0.001). (E) A shift in the Vhalf values of activation and inactivation was also observed for NaV1.7 where Vhalf values for activation of 433 cells in standard internal were significantly more negative compared with 29 cells in fluoride-free internal and the Vhalf of inactivation for 493…

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  FIGURE 5. Pharmacology of NaV1.5 recorded in standard and fluoride-free internal solution. (A) Raw current traces from an example CHO cell expressing NaV1.5 recorded in standard internal solution were blocked by tetracaine using a 3-step protocol (voltage protocol shown at the top). Traces elicited by the first (holding potential –120 mV) and second (holding potential –100 mV) step are shown. (B) Raw current traces from an example CHO cell expressing NaV1.5 recorded in fluoride-free internal solution were blocked by tetracaine. (C) Corresponding time course of block by tetracaine in standard internal solution for an average of 23 wells (at different holding potentials), time course in DMSO for an average of 41 wells is also shown. (D) Corresponding time course of block by tetracaine in fluoride-free internal solution for an average of 10 wells (at different holding potentials), time course in DMSO for an average of 35 wells is also shown. (E) Concentration response curves for tetracaine in standard internal solution or fluoride-free solution at a holding potential of –120 mV are shown overlaid. (F) Concentration response curves for tetracaine in standard internal solution or fluoride-free…

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  FIGURE 6. Activation by internal free Ca2+ of KCa3.1 recorded in standard and fluoride-free internal solution. (A) CHO cells expressing KCa3.1 were captured with almost 50% success rate for RSeal ≥ 1 GΩ with standard and in fluoride-free internal solution (left). If a QC cutoff of RSeal ≥ 0.25 GΩ was used, the success rate was increased in both conditions (right). Similar success rates were also achieved with multi-hole (4X) chips (gray). (B) Raw current traces from example CHO cell expressing KCa3.1 recorded in both standard and fluoride-free internal solution, activated by 1 μM free internal Ca2+ and blocked by 1 mM Ba2+ (external). (C) Success rates after applying initial RSeal (>0.25 GΩ) and current QC plot of IPeak values at –120/60 mV for standard and fluoride-free solutions after internal application of 1 μM free Ca2+. (D) IT plot during application of internal free Ca2+ with 0.3, 1, and 3 μM free internal Ca2+ and subsequent block by external Ba2+ (1 mM) at –120/60 mV. (E) Current values at –120 or 60 mV in increasing concentrations of internal free Ca2+ were normalized to maximum peak amplitude and fit using a Hill equation which shows a slightly increased EC50 in standard…

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  FIGURE 7. hiPSC-CMs recorded in standard and fluoride-free internal solution. hiPSC-CMs were captured with almost 60% success rate for RSeal ≥ 0.25 GΩ when using standard internal solution and about 25% RSeal ≥ 0.25 GΩ in fluoride-free internal solution in preliminary experiments. The inset shows an example of a CaV current recorded from an example cell in standard (top, dark blue) and fluoride-free (bottom, light blue). The voltage protocol used is also shown and only response to voltage step to 0 mV is shown in the raw trace examples.