Working with OpusXpress: methods for high volume oocyte experiments

Abstract

OpusXpress is a semi-automated system for high throughput voltage clamp recording from Xenopus oocytes. We participated in the development process for this system and were the only laboratory to field test a prototype. Subsequently, we obtained an early production model that we have used on a regular basis for the last seven years, conducting many thousands of experiments, publishing extensively, and carrying out collaborative research in drug discovery. In this article, we relate our experience with the OpusXpress recording system and large volume oocyte handling. We provide our standard operating procedures and outline the organization of our successful team. Some of our advice is specific to researchers fortunate enough to have access to an OpusXpress system, but most of it is applicable to any group using Xenopus oocytes for the heterologous expression of ion channels.

Figure 1

Shown on the left are preproduction images for the OpusXpress headstage and electrode arrays relative to the recording chamber. On the right are photographs of the OpusXpress that has been in service in the Papke laboratory since 2002. In the upper pictures one of the headstage-electrode units has been raised up, as it would be to change electrodes. In the middle pictures the Chamber 1 voltage headstage-electrode unit has been lowered and has begun to move toward the chambers. The bottom pictures show the final approach of the headstage-electrode unit toward one set of four recording chambers. Note that the final chamber design shows several notable modifications/refinements relative to the pre-production drawing. The electrodes point to the position for the oocyte placement. Above that point is the inflow for the bath solution and the round depression for pipette tip solution delivery. Solution delivered to the chamber flows in both directions around the cell to the outflow tube at the bottom of the chamber.

Figure 2

OpusXpress in action. A) The flight deck: the “V8” array of chambers is in the center. Behind the chambers to the left is the peristaltic pump for bath outflow and the syringe pumps for the pipette delivery system. To the right of the V8 are the racks for drug well plates and pipette tips, and furthest right is the bin for used tips. The video camera arm is in the rear, in its “homed” position. B) The two peristaltic pumps for the bath buffers. Tubes for the eight “buffer A” lines are in a single reservoir beaker. Each line has a replaceable filter at the end. C) One of the computer monitors with a chamber in view on the camera window. D) Drug wells and pipette tips in place. E) Aspiration of drug solutions. F) Movement of the tips toward the chambers. G) Drug delivery. H) Disposal of used tips.

Figure 3

Sample experiment sheet for an ACh concentration-response study of muscle-type (α1β1γδ) nAChR conducted on October 28, 2009 using cells injected on October 27, 2009 and an ACh stock solution made up the same day as the experiment. The ACh controls were 30 μM and the test solutions ranged for 100 nM to 300 μM. The initials (RLP) indicate that the data were analyzed by the first author.

Figure 4

The loaded drug wells for the experiment described by the data sheet in Figure 3. A) The loading of the wells for a single cell. Control solutions are green, experimental solutions are in graduated shades of red. Note that this experiment utilized only 18 out of the possible 24 steps. B) The loading of the two-plate set to provide drug delivery for all eight cells.

Figure 5

Sample traces from α4β2 nAChR expressing cells, viewed in Clampfit. A) The slow decay of these responses indicated to the users that the flow dynamics were suboptimal. B) Sample traces from α4β2 nAChR expressing cells obtained after the tubing was replaced in the perfusion system.