Protocol

Calibration of Micropipette Tips

This protocol was adapted from "Microinjection of Fluorophore-Labeled Proteins," Chapter 5, in Live Cell Imaging (eds. Goldman and Spector). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

INTRODUCTION

This protocol describes an easy method for calibrating micropipette tips that have been pulled in the laboratory. It is essential to estimate the internal diameter of the pulled micropipette tip when adjusting parameters for a new puller or new type of glass tubing. A tip diameter of ~0.3 µm is optimal for the microinjection of mammalian cells in culture (e.g., CHO, PtK1, and COS-7). A 10% increase in diameter increases the delivery rate by more than 30% and can cause cell damage. A smaller tip diameter will result in frequent clogging from protein aggregates.

RELATED INFORMATION

The method described here is a modification of a protocol by Hagag et al. (1990), which describes how to use a Leitz micromanipulator.

MATERIALS

Reagents

Methanol

Equipment

Air pressure regulator (e.g., model IM-200, Narishige)

Inverted microscope

Micromanipulator (e.g., Leitz)

Micropipette holder

Micropipettes to be tested

METHOD

1. Fill a 35-mm culture dish with methanol.

2. Place the holder into the micromanipulator.

3. Attach the measuring micropipette tightly to a holder that is connected to the air pressure regulator.

4. Place the culture dish on the microscope stage.

5. Turn on the air pressure regulator.

6. Set up the microinjection time for 1 minute.

7. Turn on the compressed air or nitrogen gas.

8. Position the tip of the micropipette over a dish filled with methanol.

9. Lower the tip of the micropipette into the methanol. An objective need not be used to observe bubbles coming out from a tip. However, observation with a low-magnification objective may be useful.

10. Push the "injection" button.

11. Watch for bubbles. If bubbles do not appear, increase the injection pressure until bubbles begin escaping from the tip of the micropipette.

12. Record the minimal pressure at which bubbles appear. The pressure is shown on the display in kiloPascals (kPa).

13. Substitute the recorded pressure into the equation below to determine the internal micropipette tip diameter:

diameter (µm) = 70.4/[pressure (kPa)]^1.01.

REFERENCES

Hagag N., Viola M., Lane B., Randolph J.K. 1990. Precise, easy measurement of glass pipet tips for microinjection or electrophysiology. BioTechniques 9: 401–406.[Medline]