Protocol

Microinjection of Protein Samples

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 a method for microinjecting proteins into the nucleus or cytoplasm of adherent cells. Microinjection equipment can be obtained from a number of suppliers; this protocol has been used with the Narishige IM-200 air pressure regulator and the Leitz micromanipulator. Using this system, it is possible to microinject a constant volume within a 50% difference among cells.

MATERIALS

Reagents

Cells for microinjection

Fluorescent-labeled dextran of high molecular weight, 5 mg/ml (Sigma) (optional; for practicing microinjection).

Fluorescent-labeled dextran is a useful practice material because it can be observed immediately after microinjection; it demonstrates whether material is flowing properly out of the pipette tip, and it remains in the cell compartment into which it is injected (i.e., cytoplasm or nucleus).

Protein sample(s) for microinjection

Equipment

Air pressure controller (model IM-200, Narishige)

Capillary tubing for micropipette fabrication

Borosilicate glass is well-suited for most applications.

Coverslips, etched (Bellco Glass)

Etched coverslips can also be prepared in the laboratory, as described in Plating Cells for Microinjection.

Coverslips can be attached to a 35-mm dish with a 20-mm diameter hole in the bottom, using either vacuum grease or hypotoxic silicone glue (e g., Sylgard 184, Dow-Corning). Use of vacuum grease allows easy removal of the coverslip from the dish. Dishes with attached coverslips can be sterilized by UV illumination for 30-45 minutes prior to cell plating.

35-mm culture dish with a 20-mm diameter hole in the bottom

Vacuum grease or hypotoxic silicone glue (see the note above)

Inverted microscope with desired optics (e.g., DIC or phase contrast)

A combination of fluorescence observation and microinjection requires plan achromatic objectives (Plan-Fluor or Plan-Fluotar).

Micromanipulator (Leitz)

Micropipette holder

The size of the holder should match the outer diameter of capillary tubing in order to hold the micropipette tightly.

Pipette puller (e.g., David Kopf Instruments)

Pipette storage container (e.g., World Precision Instruments)

Pulled micropipettes can also be stored in a flat dish with a lid. Attach micropipettes to the bottom with a loop of double-stick tape to keep the tips elevated.

Pressure source (nitrogen tank or compressed air from the house supply)

Compressed air can be used only for injection of oxygen-insensitive materials.

Vibration isolation table (e.g., Newport Corp. or Technical Manufacturing Co.)

METHOD

1. At least 12 hours prior to microinjection, plate cells into a dish with a photoetched coverslip in the bottom.
Most cell types are very sensitive to plasma membrane damage during the early hours after replating; 24 hours from plating to microinjection is optimal.

2. Pull micropipettes following puller manufacturer’s recommendations and store them in a container.
A tip diameter of ~0.3 µm is optimal for the microinjection of mammalian cells in culture.
Micropipettes are usable for a couple of days, but no longer than 1 week.

3. Optional: Calibrate the diameter of tips as described in Calibration of Micropipette Tips.

4. Prepare protein sample (or fluorescent-labeled dextran) for microinjection as described in Preparation and Loading of Protein Samples for Microinjection.

5. Set up the dish with cells on the microscope stage and find a suitable cell for injection. Always choose healthy-looking cells.

6. Use the printed grid pattern to record the location of cells to be microinjected. (See Troubleshooting)

7. Turn on the air pressure controller.

8. Turn on compressed air or nitrogen gas.

9. Attach the holder to the micromanipulator.

10. Load the micropipette with protein to be microinjected, as described in Preparation and Loading of Protein Samples for Microinjection.
Perform steps from this point onward rapidly to avoid drying of the sample at the tip and clogging of the micropipette.

11. If not done during loading, attach the micropipette tightly to the holder connected to the air pressure controller.

12. Use the "balance pressure" knob to apply a small amount of pressure to the micropipette before lowering it into the dish to prevent inflow of culture medium into the micropipette caused by capillary action. (See Troubleshooting)

13. Position the tip of the micropipette at a 30°-40° angle to the microscope stage.

14. Lower the tip into the medium.

15. Locate the needle just above the center of the objective lens.

16. Find the micropipette tip under the microscope.

i. First find the shadow from the tip. The shadow will not be seen without performing the lateral movements of the micropipette.
Optional: Shift to the low-power objective while guiding the micropipette. The wider the field of view is, the easier to find the tip.

ii. Use the coarse focus knob to slowly lower the micropipette tip and move the tip transversely (along the y axis) with a small amplitude. The tip gives two shadows when it is above the focal plane (cells should be in focus), one from each side of the tip wall. The higher the tip, the larger the distance between the two shadows.

iii. Lower the micropipette with the fine adjustment until the tip is clearly seen just above the cells. From time to time, move the micropipette sagittally (along the x axis). This is needed to maintain the tip in the center of the field. (See Troubleshooting)

17. Optional: Check the flow before microinjection. Focus on the tip of the micropipette. Check whether small particulates are being blown from the tip into the dish. If a fluorescent-labeled protein is microinjected, the material flowing out of the pipette can be seen by looking in the appropriate fluorescent channel. (See Troubleshooting)

18. Move the needle tip over the top of the cell that will be microinjected.

19. Increase the pressure to 5-12 kPa to generate a continuous flow from the micropipette tip. When the tip touches the cell and penetrates the cortex, the solution continues to enter the cell until the micropipette is pulled out. (See Troubleshooting)

20. Prepare to microinject the sample.
For microinjection into the cytoplasm:

i. Position the needle tip above the thick part of the cytoplasm (usually thicker near the nucleus).

ii. Lower the tip by fine control and penetrate the cell by gently touching the cell cortex.
If injection occurs, a "wave" will be seen spreading from the point of injection. If the cell does not react, raise the needle and increase the pressure. Try microinjection of another cell. Do not repeatedly penetrate the same cell.

For nuclear microinjection:

i. Position the needle tip above the nucleus.

ii. Lower the tip by fine control and penetrate the cell (more deeply than for cytoplasmic injection). The tip has to enter the cell nucleus.

If injection occurs, a bright zone will appear around the tip. (See Troubleshooting)

21. Microinject the selected cells. Keep the microinjection process to less than 30 minutes.
Some cells are very sensitive to pH changes occurring while a dish is outside of the CO₂ incubator. They cannot be microinjected for longer than 15 minutes unless special bicarbonate-free medium is used. (See Troubleshooting)

22. Return the dish to the incubator.

23. Leave cells in the incubator at least 30 minutes before continuing microinjection.

TROUBLESHOOTING

Problem: Cells were grown to a semiconfluent monolayer, but the grids and letters cannot be seen very well on the coverslip.

[Step 6]

Solution: When the coverslip was attached, it was placed with the grid facing up. Cells mask the grid pattern if both are in the same focal plane. The grid can be seen only in areas that are free from cells. To relocate the cells in a semiconfluent monolayer, mount the coverslip such that the grid and cells are located on opposite sides of the coverslip.

Problem: The coverslip grid can be seen using the dry objective, but not when using the oil objective.

[Step 6]

Solution: The grid is facing down into the oil that obscures the grid pattern. Remove the oil from the coverslip with absolute ethanol and find the cells using the dry objective. Do not move the dish, then change the objective.

Problem: The micropipette is blown out of the holder when pressure is applied.

[Step 12]

Solution: The micropipette was not attached tightly to the holder, or the size of the holder does not match the size of the outer diameter of tubing used for micropipette fabrication.

Problem: The micropipette tip cannot be found.

[Step 16]

Solution: The micropipette is not in the field of observation. Try to place the micropipette directly above the center of the objective lens. Use the low-power objective to guide the micropipette. Make sagittal and transverse displacements of the micropipette with greater amplitude. Try to view the shadow. Lower the micropipette slightly and repeat the procedure.

Problem: The micropipette tip breaks while it is being located under the microscope.

[Step 16]

Solution: Move the micropipette transversely to place the end of the shadow into the center of the field. When the needle is lowered, the tip should be directly in the center. If the shadow cannot be seen clearly when the needle is lowered, the tip may touch the coverslip away from the field of observation.

Problem: There is no flow from the micropipette. Particles cannot be seen coming out from the tip.

[Step 17]

Solution: The tip of the micropipette might be clogged by aggregates in the sample. Raise the needle and briefly press the "clear" pushbutton on the air controller. This will apply high pressure, which may clear the clogged pipette. If this procedure fails, try fabricating tips with larger openings (decrease filament current) or centrifuge the sample at high speed. It is also possible that the filled tip was left too long in the air before dipping it into medium, allowing the sample to dry at the tip and clogging the micropipette. Other factors that can impede flow are bubbles in the tip or micropipettes that become sealed during the pulling process (seeTroubleshooting steps below).

Problem: There is a bubble at the tip of the micropipette.

[Step 17]

Solution: If bubbles occur frequently, try loading samples from the tip ("front-loading") as described in Preparation and Loading of Protein Samples for Microinjection.

Problem: The micropipette appears to be sealed.

[Step 17]

Solution: It is possible to break a sealed or clogged tip by placing the "sealed" tip above a field that is free of cells. Lower the tip very slowly until a small piece of the tip is broken off. Then try to inject the cells again. When preparing tips in the future, decrease the current for pipette fabrication.

Problem: The loaded micropipette becomes empty when pressure is increased.

[Step 19]

Solution: The micropipette tip has too large an opening. This may be due to a broken micropipette tip or a micropipette with too large an internal diameter.

Problem: The wave can be seen when microinjecting the material into the cell, but the bright fluorescence cannot be seen inside the cell.

[Step 20]

Solution: The low balance pressure was not applied before dipping the tip into the dish. Medium was drawn into the tip by capillary action and, thus, mostly medium was microinjected.

Problem: Micropipette clogs after microinjecting only a few cells.

[Step 21]

Solution: Increase the pressure from time to time during microinjection. Use the "clear" feature of the gas regulator to unclog the tip. Centrifuge the sample longer.

Problem: Cells explode during microinjection.

[Step 21]

Solution: The flow from the micropipette tip is too high. This may be due to too high a pressure being used for microinjection; if so, decrease the pressure. Another possibility is that the internal diameter of the tip is too large; if so, increase the current when pulling the micropipette.

Problem: Cells are known to be pH sensitive and/or they do not appear to tolerate time outside the CO₂ incubator.

[Step 21]

Solution: Leibovitz’s L-15 medium (GIBCO-Invitrogen) can be used for microinjection of pH-sensitive cells (e.g., LLCPK). Microinjection of primary neuronal cells can be done using L-15 medium enriched with glucose (0.5%) and L-glutamine (0.9%). Medium without bicarbonate and phenol red, but containing 20-25 mM HEPES, can be used for cells that cannot tolerate L-15 medium.

Problem: Microinjection of neuronal cells was tried and all cells died.

Solution: Begin practicing the microinjection technique with CHO or COS-7 cells, which are more tolerant to microinjection. Practice for a week or two before attempting microinjection of more sensitive cells such as neurons. Do not use a freshly plated culture of neuronal cells for microinjection. Instead, primary neurons, cultured for 1 day, are recommended to start with. About 50% survival is a good result for neuronal cells.

Problem: Very high percentage of cell death occurs after microinjection. The cells permanently change morphology and die even though too much sample was not microinjected.

Solution: The cells that are microinjected may be very sensitive. Equipment modifications that might improve cell survival include changing the tubing used for micropipette fabrication, using tubing with a smaller outer diameter, fabricating very fine tips, and using lower pressure for microinjection. It is also possible that the probe is toxic for the cells. Check whether the original protein stock (if commercial) contains any toxic component (glycerol, azide, etc.) for protein stabilization. Try to microinject buffer alone and see if the cells show the same percentage of survival. If the buffer itself contains the toxic component, the material for microinjection should be dialyzed into the appropriate carrier solution (buffer for microinjection).

Problem: How does one know whether the cells are dying?

Solution: Check whether the cells change morphology gradually after microinjection. Usually, good microinjection does not cause permanent changes in morphology and cells recover within 5-10 minutes. The first sign that cells are dying is that mitochondria become condensed and exhibit high contrast (when viewed using phase-contrast optics).