Microinjection is a very common technique used in cell and molecular biology labs. It is used to penetrate the cell or nuclear membrane in order to introduce genetic material, drugs, fluorescent labels, etc. Various cell types require tips with variable dimensions and shapes. In many cases the glass tips can be pulled using a glass puller and microloader pipette tips can be obtained by heating and filing a normal pipette tip. However, quality-controlled manufactured products ensure the best possible performance.
Eppendorf® Femtotips® and Microloader Pipette Tips enable rapid, precise, and highly reproducible microinjections. The Eppendorf® Femtotips® are sterile, individually wrapped and quality control tested. They come in various diameters (inner and outer) and have a precise length accuracy and a screw thread for rapid mounting in the micromanipulator. To ensure a perfect seal, I use an extensible tape wrapped around the screwed thread. Once the tip is prepared, I screw it in a MMN-1 coarse manipulator that also has a MMO-202 N three dimensional joystick-controlled hydraulic micromanipulator system from Narishige . I visualize the preparation for injection with a Nikon TE 2002-E microscope.
The Femtotips® can be used for microinjection capillary for reproducible injection into adherent or suspension cells. I used Femtotips® with an opening of 0.5 µm (inner diameter) and 0.7 µm (outer diameter), (±0.1 µm). In most cases, I microinjected Drosophila embryos aligned on a glass plate and I needed to break the tip in order to allow the solution to flow into the embryos. To break the tip in a controlled way, I first filled the tip with the solution needed to be injected and then rubbed the tip on the edge of a glass slide while applying pressure. Once a drop of liquid was seen coming from the tip, the tip was ready for injection. If the solution to microinject precipitates or has a higher viscosity or if Drosophila embryo content aspirates into the tip, the tip blocks. To avoid this complication, it is advisable to briefly spin the solution before microinjection. Another problem associated with glass needles for microinjection is that (especially during initial training in microinjection) the frequency of breaking is quite high. If money is an issue in the lab, it is better to use manually pulled needles at the beginning in order to become familiar with making the prep, finding the tip, adjusting the position of the needle, loading the tips and initial microinjections. Once a level of proficiency has been obtained, then go to the Eppendorf tips for real injections.
Microloader Pipette tips are used to fill microinjection capillaries. They come in a sterile box that can be resterilized. The filed tip is very fragile and must be carefully inserted in the glass needle as to avoid bending which can obstruct it. Also, it is advisable to introduce the filler to the very tip of the needle and then to dispense the liquid in order to avoid the formation of air bubbles that can make the injection very difficult.
I have used Eppendorf® Femtotips® and Microloader Pipette Tips extensively to microinject Drosophila embryos with various proteins (RanT24N, RCC1, importins alpha and beta, RanGAP, CAS), fluorescently labeled proteins (rhodamine actin, rhodamine tubulin, fluorescein actin), antibodies (anti-aurora A, anti-peanut) and drugs (colcemid).
Postdoc
Department of Medical Genetics and Microbiology
University of Toronto