Method for Rapid Enzymatic Cleaning for Reuse of Patch Clamp Pipettes: Increasing Throughput by Eliminating Manual Pipette Replacement between Patch Clamp Attempts

Abstract

The whole-cell patch-clamp method is a gold standard for single-cell analysis of electrical activity, cellular morphology, and gene expression. Prior to our discovery that patch-clamp pipettes could be cleaned and reused, experimental throughput and automation were limited by the need to replace pipettes manually after each experiment. This article presents an optimized protocol for pipette cleaning, which enables it to be performed quickly (< 30 s), resulting in a high yield of whole-cell recording success rate (> 90%) for over 100 reuses of a single pipette. For most patch-clamp experiments (< 30 whole-cell recordings per day), this method enables a single pipette to be used for an entire day of experiments. In addition, we describe easily implementable hardware and software as well as troubleshooting tips to help other labs implement this method in their own experiments. Pipette cleaning enables patch-clamp experiments to be performed with higher throughput, whether manually or in an automated fashion, by eliminating the tedious and skillful task of replacing pipettes. From our experience with numerous electrophysiology laboratories, pipette cleaning can be integrated into existing patch-clamp setups in approximately one day using the hardware and software described in this article. Graphic abstract: Rapid enzymatic cleaning for reuse of patch-clamp pipettes.

Keywords: Automation; Electrophysiology; Enzymatic detergent; High-throughput; Patch-clamp.

Figure 1.. Pipette cleaning methods.

A. Process flow chart for traditional manual patch clamping without pipette cleaning. Removing, filling, and installing fresh pipettes takes between 60-120 s. B. Process flow chart for manual patching with automated cleaning (“push-to-clean”). Automated cleaning can be run in as little as 30 s. C. Close up images of pipette being moved from the experimental chamber (left) to the cleaning bath (middle) and to the rinse bath (right) before returning to the experimental bath to patch another cell. Scale bar is 25 mm. D. Custom experimental chamber for pipette cleaning featuring fluid inlet and outlet, inset for ground wire, and external baths for cleaning and rinsing solutions. Scale bar is 1 cm.

Figure 2.. Improvements to pipette cleaning.

A. Each trace represents the number of whole-cell recordings in HEK 293 cells as a function of the number of recording attempts with a single pipette. Each trace is the average of at least three pipettes. The “Saline” trace is a negative control (i.e., cleaning solution replaced with extracellular solution), and a 100% theoretical maximum is included for reference. The “Alconox” trace shows the performance of 2% w/v Alconox cleaning, which decreases as a function of the number of attempts. The “Tergazyme” trace shows no decrease in yield for 30 attempts with 2% w/v Tergazyme. The “Optimized” trace represents 2% w/v Tergazyme cleaning with optimized pipette positioning relative to the cell surface for gigasealing. The “Tergazyme” performance is superior to that of Alconox (*, P = 1.375E-5, Kolmogorov-Smirnov test). The “Optimized” performance is superior to that of “Tergazyme” (**, P = 0.04368, Kolmogorov-Smirnov test). B. Success rate of whole-cell patch-clamp as a function of the number of cleans using 2% w/v Tergazyme shows no significant decrease in likelihood of subsequent whole-cell recording (Odds ratio (OR) = 1.0067, CI: 0.97-1.04, P = 0.69, n = 215 attempts, each attempt is for n = 7 pipettes, except attempts 29 and 30, which are for n = 6). C. Optimized indentation with Tergazyme cleaning shows no significant decrease in likelihood of subsequent whole-cell recording (OR = 1.00, CI: 0.94-1.06, P = 0.95, n = 124 attempts, each attempt is for n = 4 pipettes).

Figure 3.. High-throughput opsin screening with pipette cleaning.

A. Yield curve for a single pipette channelrhodopsin-2 (ChR-2) characterization experiment (46/51 attempts, 90% yield). B. Representative photocurrent trace (voltage clamp) in response to an initial pulse of 500 ms 480 nm LED pulse recorded from transiently transfected HEK 293 cell showing a large peak photocurrent response. C. Photocurrent traces (voltage clamp) from cells recorded in the middle of a series of 500 ms light pulses showing steady-state photocurrents over many pipette cleans. Large initial photocurrent responses are typical of ChR-2 (B) and are reduced in subsequent stimulation pulses (C) ( Lin et al., 2009 ).

Figure 4.. Upper limits of pipette cleaning with 2% w/v Tergazyme.

A. Yield curves for individual pipettes showing cleaning for over 90 recording attempts with associated failure modes. The “success” trace shows effective pipette cleaning, the “clog” trace shows reversible pipette tip clogs that cause low yield over time, and the “break” trace shows experiments terminated by broken pipette tips. Theoretical maximum (100% yield) included for reference. B. Representative pipette images taken at 40× magnification for each failure mode in (A). Scale bar is 1 µm. C. Individual gigaseal resistance traces from the “success” trace (n = 122 gigaseal attempts). D. Access resistance of cells recorded in the “success” trace (n = 101 whole-cell recordings).

Figure 5.. Tergazyme cleaning is effective without a rinsing step in acute mouse brain slices.

A. The success rate of patching without a rinse step does not decrease significantly with the number of cleans (OR = 1.14, CI: 0.87-1.41, P = 0.34, n = 36 attempts). The number of pipettes used for each experiment is noted above each number of reuses. B. Yield plot for a single pipette using 2% w/v Tergazyme without rinsing. C. Representative recordings of evoked action potential firing in current clamp from three neurons recorded with a single pipette cleaned in 2% w/v Tergazyme without rinsing. D. Enlarged single evoked action potentials from the neurons in (C).

Figure 6.. Calibration of pipettes for pipette cleaning.

Positions used by the push-to-clean software for each cleaning attempt. Images in (A) refer to saved position values in (B). Briefly, (1) refers to position directly above target cells or tissue, (2) refers to a z-location above the clean and rinse baths, (3) refers to the position where the tip is submerged in cleaning solution, and (4) refers to the position where the pipette tip is submerged in the rinsing solution.