Localized Sampling of Oocyte Cytoplasm

3.1.1. Oocyte Preparation

1. Surgically remove stages V and VI oocytes from X. laevis frogs.

2. Isolate individual oocytes after collagenase digestion (13).

3. Maintain oocytes at 18°C in ND96 until use.

4. Make a 0.5-mm "V"-shaped depression within a shallow (1-2 mm) reservoir created in a block of electrically nonconductive material such as delrin. The shallow reservoir should be contiguous with a deeper reservoir of 1 to 2 mL total volume (Fig. 2).

5. In preparation for sampling of oocyte cytoplasm into a capillary, place a single oocyte into the "V"-shaped depression (see Note 2).

3.1.2. Construction of Capillary Sampling Apparatus

For aspiration of sample volumes in the picoliter range, a capillary possessing a sharpened tip (see Subheading 3.1.2., step 1) can be inserted and withdrawn from the oocyte. Coring action extracts a sample proportional to the depth of insertion (9). For rapid, computer-controlled insertion and extraction, the capillary is attached to the end of a piezoelectric motor element with a custom-made mount (Fig. 3).

1. Etch the tip of the capillary to a sharp tip with concentrated hydrofluoric acid (HF). Appropriate caution must be taken as the HF solution is highly caustic, and all steps

Fig. 1. Schematic representation of the equipment to measure [IP3]. The stage of an upright microscope contains the custom-built delrin holder with a "V"-shaped depression to hold the Xenopus oocyte (XO). A microscope objective (MO) is used for viewing the oocyte and positioning the sampling tip (ST). A micromanipulator (M) on the stage of the microscope holds the sampling tip. The micromanipulator is mounted on a rail so that it can be translated and readily removed from the stage. The sampling tip is spliced into a "T" junction connected to the capillary outlet (CO) and a side port (SP) capillary. The side port capillary is attached to a vacuum valve (VV). The vacuum valve power supply (VVPS), the capillary electrophoresis power supply (CEPS), and the sampling power supply (SPS) are controlled through a data input/output board in a personal computer (PC). The capillary outlet is placed in the outlet reservoir (OR) on the stage of an inverted microscope (IM). The outlet reservoir is made from an "O" ring cemented to a cover slip on which the IP3-detector cell (DC) is plated. The fluorescence of the detector cell is measured with a photomultiplier tube (PMT) or intensified CCD camera. The fluorescence signal from the PMT is digitized by an A/D board and monitored, and the data are stored in the personal computer.

Fig. 1. Schematic representation of the equipment to measure [IP3]. The stage of an upright microscope contains the custom-built delrin holder with a "V"-shaped depression to hold the Xenopus oocyte (XO). A microscope objective (MO) is used for viewing the oocyte and positioning the sampling tip (ST). A micromanipulator (M) on the stage of the microscope holds the sampling tip. The micromanipulator is mounted on a rail so that it can be translated and readily removed from the stage. The sampling tip is spliced into a "T" junction connected to the capillary outlet (CO) and a side port (SP) capillary. The side port capillary is attached to a vacuum valve (VV). The vacuum valve power supply (VVPS), the capillary electrophoresis power supply (CEPS), and the sampling power supply (SPS) are controlled through a data input/output board in a personal computer (PC). The capillary outlet is placed in the outlet reservoir (OR) on the stage of an inverted microscope (IM). The outlet reservoir is made from an "O" ring cemented to a cover slip on which the IP3-detector cell (DC) is plated. The fluorescence of the detector cell is measured with a photomultiplier tube (PMT) or intensified CCD camera. The fluorescence signal from the PMT is digitized by an A/D board and monitored, and the data are stored in the personal computer.

should be performed in a chemical fume hood. Etching is performed as follows: A 1-cm region of polyimide coating is burned from the capillary at a point 5 cm from one end and cleaned with ethanol to expose the fused silica. The capillary is connected to a nitrogen tank and purged with 10 psi of nitrogen to prevent diffusion of HF into the capillary. The capillary is threaded through a 1000-|lL disposable pipet tip (e.g., Rainin Pipetman P-1000) containing 200 |L HF. The region of exposed fused silica is positioned within the HF solution, and the capillary is fixed in place on its short end. The other end of the capillary is weighted with a small weight (35 g) and allowed to hang from the pipet tip. The HF will etch the exposed glass until the weighted capillary breaks free (~20-30 min). On completion of the etching process, the capillary will typically be sharpened to a tip retaining the original inner diameter of the lumen, but which now possesses a 2- to 5-|m wall.

2. Position the piezoelectric-mounted capillary adjacent to the oocyte using a three-axis micromanipulator. Typically, the tip of the capillary is placed at the upper pole of the oocyte, very nearly touching the cell membrane.

Fig. 2. Diagram of the custom-made delrin oocyte holder (DH) and reservoir. (A) Side view and (B) top view of the holder for the Xenopus oocyte (XO) and inlet buffer reservoir (IR). The "V"-shaped depression drilled in the holder is shown on the right (H). The deeper section of the inlet buffer reservoir is shown on the left. The microscope stage is modified to rigidly maintain the holder in place. The dimensions of the oocyte holder are as follows: 4.5 cm high, 4 cm wide, and 5 cm long.

Fig. 2. Diagram of the custom-made delrin oocyte holder (DH) and reservoir. (A) Side view and (B) top view of the holder for the Xenopus oocyte (XO) and inlet buffer reservoir (IR). The "V"-shaped depression drilled in the holder is shown on the right (H). The deeper section of the inlet buffer reservoir is shown on the left. The microscope stage is modified to rigidly maintain the holder in place. The dimensions of the oocyte holder are as follows: 4.5 cm high, 4 cm wide, and 5 cm long.

Fig. 3. Diagram of the sampling tip. The custom-built delrin holder (DH) with a "V"-shaped hole holds the Xenopus oocyte (XO) in the inlet buffer reservoir (IR). The sampling tip (ST) is cemented to the ends of a glass sleeve (GS). A nylon screw (S) is used to hold the glass sleeve in place in the custom-built clamp (CBC) connected to an "x-y-z" micromanipulator (M) through a piezoelectric device (PED). The oocyte and sampling tip are imaged through the microscope objective (MO).

Fig. 3. Diagram of the sampling tip. The custom-built delrin holder (DH) with a "V"-shaped hole holds the Xenopus oocyte (XO) in the inlet buffer reservoir (IR). The sampling tip (ST) is cemented to the ends of a glass sleeve (GS). A nylon screw (S) is used to hold the glass sleeve in place in the custom-built clamp (CBC) connected to an "x-y-z" micromanipulator (M) through a piezoelectric device (PED). The oocyte and sampling tip are imaged through the microscope objective (MO).

3. At the desired time, insert the sharpened capillary tip into the oocyte to the desired depth (60-300 |lm), followed by withdrawal of the tip into the solution surrounding the oocyte using the computer-controlled piezo element. The buffer solution serves as the buffer for electrophoresis (see Note 3).

4. Initiate electrophoresis.

5. Apply a vacuum to the capillary during the insertion to aspirate nanoliter volumes of cytoplasm if desired. This aspiration is accomplished by attaching a valve-operated vacuum line to the capillary at a position 7 cm from the capillary inlet. The inlet capillary should be of 75-|m id and 7 cm long to improve sample aspiration. This capillary is attached to the zero dead volume "T" junction. The separation capillary (50-|m id, 360-|m od, 45 cm long) is connected to the "T" on the straight-through position corresponding to the inlet capillary. To connect the vacuum line to the junction, a capillary (100-|m id, 360-|m od, 5 cm long) is attached to the perpendicular third leg of the "T." A length of Teflon tubing (2 cm, 0.38-mm id) is connected to the capillary, and is cemented to the common outlet of the three-way valve. The normally open inlet of the valve is open to atmospheric pressure; the normally closed inlet is connected to a vacuum (709 mmHg). To remotely open the valve for application of a vacuum to the inlet capillary during the sampling procedure, a relay switch is interposed between the valve and a 12-V power supply. This relay is controlled by the personal computer PC via the A-to-D board.

6. Perform the cytoplasmic sampling. This is accomplished using a customized software program written to control the piezoelectric motor, the valve, time delays between piezoelectric motor movement and valve opening and closing, and the initiation of electro-phoresis. After positioning the capillary tip adjacent to the desired sampling location (see Subheading 3.1.2., step 2), a series of software-controlled events are triggered: (1) movement of the piezoelectric motor downward, driving the capillary tip into the oocyte; (2) a variable time delay; (3) opening of the vacuum valve, causing aspiration of cytoplasm into the capillary; (4) closing of the vacuum valve; (5) another time delay; (6) upward movement of the piezoelectric element, withdrawing the capillary tip from the oocyte; (7) a third time delay; and (8) initiation of electrophoresis by application of a voltage across the capillary. Typical time delays are 100 ms.

3.1.3. Determination of the Volume of Cytoplasm Sampled

The detector cell acts primarily as a detector of the mass of IP3 in the sample rather than its concentration (6). Because the heterogeneity of the oocyte cytoplasm can lead to significant variation in the volume sampled, it becomes imperative to accurately quantify the sample volume for each measurement to determine the IP3 concentration (6,9). This is accomplished by loading a radioactive marker into the oocyte prior to sampling.

1. Load the oocyte with [a-32P] ATP (50 nL, ~105 cpm) 15 to 30 min prior to experiments to allow adequate time for diffusion of the injectate throughout the oocyte.

2. After sampling (see Subheading 3.1.2., step 3), immediately transfer the oocyte to a liquid scintillation vial.

3. After electrophoresis, flush the capillary with buffer and collect this buffer in a second scintillation vial. The [a-32P] ATP migrates more slowly than IP3 and is retained in the capillary. Thus, the buffer flushed from the capillary contains that portion of the [a-32P] ATP sampled from the oocyte.

4. Determine the sample volume. Assuming an oocyte cytoplasmic volume of 0.5 ||L (14), the volume sampled is determined by the following equation:

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