The InjectMan® NI 2¹ is a menu-controlled, programmable micromanipulator, especially suitable for microinjection into adherent cells. The electronic connection of the InjectMan NI 2 and the FemtoJet² or FemtoJet® express ensures a very rapid and reproducible microinjection. The semiautomatic microinjection allows a coordinated process of manipulation and injection. The exact axial injection movement ensures a minimum mortality rate, and its high speed facilitates the penetration of rigid structures. Applications include semiautomatic microinjection into adherent cells including serial microinjection into fish larvae, insect embryos, etc.

Can the injection angle be varied to suit different applications (e.g. DNA injection into the gonads of worms)?Yes. The axial angle can be adjusted for semi-automatic injection and for manual injection with the axial function (e.g. into C. elegans). The adjustable angles are found in the operating manual and vary between 35° and 55° (positioned in the middle hole of the guide). For injection angles different from 45°, the angle settings must also be changed in the menu (Install/Angle).

Is it possible to connect the Transjector 5246 to the manipulators of the new generation?Yes, this is possible. For the combination of Transjector 5246 and InjectMan NI 2 you must use the following interface cable (5181 150.060).

What are the main features of the New Manipulator Generation (NMG)?- High speed for efficient penetration of rigid structures (Vmax 7,500 µm/sec) - Motors with high resolution for smooth step-free motions. - Resolution per step: 0.04 µm - Easy preset of speed or work area via control unit - Menu controlled programming - Storage of user profiles

Which working distance is necessary for an upright microscope?The minimum working distance is approx. 5 mm. In this case the capillary has to be mounted very flat, almost parallel to the table. In general, inverted microscopes are the better choice for micromanipulation experiments because of their large working distance of more than 20 mm.

How do I convert from hPa to psi?1 hPa = 0,0145 Psi 1 Psi = 68,95 hPa

What parameters are decisive for the injection volume, and how can I determine these?The injection volume depends on the set pressure and the type of capillary, as well as the residence time of the capillary in the cell. Furthermore, the injected volume depends on the type of cell and the solution to be injected. Due to the above described factors, it is not possible to give exact settings for a specific injection volume. The user always has the option of determining the injection volume for the present experiment by making a type of "calibration curve". Possible methods of determining the volume: 1. Injection of an enzyme that is normally not present in the cell (e.g. luciferase) in 50 - 100 cells and determination of the injection volume by subsequent enzyme assay: - Add pure luciferase (Sigma, final conc. 2 mg/ml) to the injection solution. - Inject an exact number of cells with the FemtoJet/FemtoJet express with a known setting. - Lyse cells and determine the luciferase activity from the extract with a luminometer (as described in the literature). - Prepare a dilution series from the injection solution (2 mg/ml) and determine the luciferase activity in this series. - Plot a curve of the measured activity versus volume. - Read off the injected volume from this curve and the measured activity of the cells. - Calculate the volume per cell from this read volume. 2. Injection of a defined radioactivity into one water drop (20 - 100 times), followed by measurement of the radioactivity (Geiger counter). The approximate injection volume can be calculated from the radioactivity of the solution. 3. Injection of fluorescence (generally coupled with a carrier protein), quantification of the injection volume by means of the detection system. When using our Femtotips, the determined settings can be retained from capillary to capillary. Typical volumes are 0.1 to 0.5 pl for injection into the cytoplasm and 0.01 to 0.05 pl for injection into the nucleus. Publications: a) Ansorge, W. and Pepperkok R., (1988): Performance of an automated system for capillary microinjection into living cells. Biochem. Biophys.Meth.16, 283-292 (Calculation by injection with fluorescence markers.) b) G. Minaschek, J. Bereiter-Hahn, and G. Bertholdt (1989): Quantification of the Volume of Liquid Injected into Cells by Means of Pressure, Experimental Cell Research 183, 434-442 (Explanation of the calculation of the injection volume depending upon pressure and time.)

What can you recommend with regard to sample preparation of the injection solution for microinjection?- Samples should always be centrifuged (for 15 minutes at maximum speed in a microcentrifuge) immediately before the capillaries are loaded. - Use the Eppendorf Microloader for filling Femtotips (from the rear). Use it only once. The liquid should be extracted from the top of the tube. Make sure that no gas bubbles are in the glass capillary. If your solution contains proteins, you should work as quickly as possible after the capillary has been loaded. If the injection solution is not introduced into the medium immediately, there is a danger of the injection solution drying in the capillary, thus blocking the Femtotips. Please find more detailed information in our Applications No. 8 "Sample preparation for microinjection", which we are happy to send you upon request.

What guidelines are available for the optimal concentration of protein or plasmid in the injection capillary?As a rule, 20-200 ng/µl DNA and 1–5 mg/ml protein are injected. With an antibody solution, the concentration should be 10 to 50 times higher than that used in the in vitro test system (e.g. Southern blot).

Do you have a publication which explains how to determine the diameter of microinjection capillaries and also indicates the influence of the diameter on the injection volume?Yes, we do have one such application: Experimental Cell Research 210, 260-267 (1994): Microinjection Technique: Routine System for Characterization of Microcapillaries by Bubble Pressure Measurement,

Is any literature available on the subject of microinjection of adherent cells using Eppendorf devices?yes, please refer to our reference database, key word InjectMan NI2, available online http://www.eppendorf.de/int/index.php?&action=library&librarynode=28820&reforder=1&refcount=10&contentid=14

Can you recommend any parameters for the injection of DNA into adherent cells?Parameters for the injection of DNA into mammalian cells using Femtotip or Femtotip II: pc = 30 to 300 hPa pi = 50 to 500 hPa ti = 0.3 to 1.5 s The parameters must be optimized according to each experiment. If you are using Femtotips, please mount the universal capillary holder at an angle of 45 °.

What is the typical injection volume?The injection volume is typically ~ 10 % of the cell volume.

Can you supply us with literature about microinjection of DNA or proteins into adherent cells?Our Application No. 8 contains interesting hints about the preparation of samples for microinjection. www.eppendorf.com

Which dextran is most suitable for microinjection?All dextrans can be used irrespective of the molecular weight. For a special application, namely compartment-specific injections, we use dextran with a molecular weight in excess of 60 kDA, as it cannot penetrate the core barrel.

Which Eppendorf devices do you recommend for the injection into insect embryos?We recommend TransferMan NK 2 or InjectMan NI 2, FemtoJet express and a footswitch for the FemtoJet express. For objects with a strong shell (Chitin) a Piezo Drill may be necessary.

Which injection pressure do I have to select for microinjection into fish embryos?Microinjections into embryos are normally performed manually with an injection pressure of 300-500 hPa and a very short injection time of 0.1-0.2 seconds. The high injection pressure prevents the injection needle from clogging.

What is the typical injection volume when injecting into drosophila embryos?The typical injection volume ranges between 60-100pl

Can you supply us with literature about microinjection into C. elegans?Yes, our Userguide No 0112/06 “Microinjection of plasmid DNA or double stranded RNA into the gonads of C. elegans” contains lots of useful information about this kind of application. The Userguide is available at our website

Can you recommend any literature about microinjection into plant cells?1. Schnorf, M. et al., An improved approach for transformation of plant cells by microinjection: molecular and genetic analysis, Transgenic Research 1, 23-30 (1991) 2. Brücker et al., Microinjection of heme oxygenase genes rescues phytochrome-chromophore-deficient mutants of the moss Ceratodon purpureus, Planta 210, 529-535 (2000)

What is important for the microinjection into plant cells?For the injection into plant cells, a pressure greater than 7,000 hPa is very often necessary. Therefore, a CellTram Oil or vario should be used for plants with a high turgor (max. pressure 20.000 hPa).

What equipment is required to inject fluorescent dyes into plant cells and to then aspirate the entire content of the cell?Since a very high pressure is required for injection into some plant cells, we recommend using the manual CellTram Oil Microinjector in combination with a micromanipulator (e.g. TransferMan NK 2), which enables material to be extracted out of the cell. If suspension cells are used, a second micromanipulator is necessary to fix the cells.

What should be taken into account when injecting proteins? .It is sometimes difficult to inject protein solutions, as any contamination can block the capillary. Therefore, the solutions must be prepared very carefully (e.g. cleaned by centrifugation columns or ultracentrifugation). Before loading the Femtotip, the solution should always be centrifuged for at least 10 minutes at the highest speed

What is the typical injection volume when injecting into drosophila embryos?The typical injection volume ranges between 60-100pl

Is it possible to control the manipulators and elctronical injectors via PC?Yes, you ca nfind a detailled description on our homepage

Which grip head is included in the delivery package of the FemtoJet/FemtoJet express?Grip head number 0. It can be used for capillaries with an outer diameter of 1,0-1,1 mm.

What is the outer diameter of the universal capillary holder?The outer diameter is 4 mm.

I want to use my own capillaries with Eppendorf microinjectors. Which capillary grip would fit my tips?All Eppendorf microinjectors (Transjector, FemtoJet/FemtoJet express and CellTram) are equipped with the Universal Capillary Holder. Different grip heads can be interchanged for the use of capillaries with different outer diameters. Use the table below to determine which grip head suits your needs. Description Capillary grip 0: fits microcapillaries with an outer diameter of 1.0 to 1.1 mm, order no. 5176 210.003 Capillary grip 1: fits microcapillaries with an outer diameter of 1.2 to 1.3 mm, order no. 5176 212.006 Capillary grip 2: fits microcapillaries with an outer diameter of 1.4 to 1.5 mm, order no. 5176 214.009 Capillary grip 3: fits microcapillaries with an outer diameter of 0.7 to 0.9 mm, order no. 5176 207.002

How can the different grip heads be distinguished?The grip heads have different numbers of notches. The grip head 0 has no notch at all; the grip head 3 has, for example, three notches

Can a PC be used to control the the New Manipulator Generation (NMG)?A PC can easily control all Eppendorf manipulators of the new generation. A detailed description can be found at our website.

Can the injection angle be varied to suit different applications (e.g. DNA injection into the gonads of worms)?Yes. The axial angle can be adjusted for semi-automatic injection and for manual injection with the axial function (e.g. into C. elegans). The adjustable angles are found in the operating manual and vary between 35° and 55° (positioned in the middle hole of the guide). For injection angles different from 45°, the angle settings must also be changed in the menu (Install/Angle).

Is it possible to connect the Transjector 5246 to the manipulators of the new generation?Yes, this is possible. For the combination of Transjector 5246 and InjectMan NI 2 you must use the following interface cable (5181 150.060).

What are the main features of the New Manipulator Generation (NMG)?- High speed for efficient penetration of rigid structures (Vmax 7,500 µm/sec) - Motors with high resolution for smooth step-free motions. - Resolution per step: 0.04 µm - Easy preset of speed or work area via control unit - Menu controlled programming - Storage of user profiles

Which working distance is necessary for an upright microscope?The minimum working distance is approx. 5 mm. In this case the capillary has to be mounted very flat, almost parallel to the table. In general, inverted microscopes are the better choice for micromanipulation experiments because of their large working distance of more than 20 mm.

How do I convert from hPa to psi?1 hPa = 0,0145 Psi 1 Psi = 68,95 hPa

What parameters are decisive for the injection volume, and how can I determine these?The injection volume depends on the set pressure and the type of capillary, as well as the residence time of the capillary in the cell. Furthermore, the injected volume depends on the type of cell and the solution to be injected. Due to the above described factors, it is not possible to give exact settings for a specific injection volume. The user always has the option of determining the injection volume for the present experiment by making a type of "calibration curve". Possible methods of determining the volume: 1. Injection of an enzyme that is normally not present in the cell (e.g. luciferase) in 50 - 100 cells and determination of the injection volume by subsequent enzyme assay: - Add pure luciferase (Sigma, final conc. 2 mg/ml) to the injection solution. - Inject an exact number of cells with the FemtoJet/FemtoJet express with a known setting. - Lyse cells and determine the luciferase activity from the extract with a luminometer (as described in the literature). - Prepare a dilution series from the injection solution (2 mg/ml) and determine the luciferase activity in this series. - Plot a curve of the measured activity versus volume. - Read off the injected volume from this curve and the measured activity of the cells. - Calculate the volume per cell from this read volume. 2. Injection of a defined radioactivity into one water drop (20 - 100 times), followed by measurement of the radioactivity (Geiger counter). The approximate injection volume can be calculated from the radioactivity of the solution. 3. Injection of fluorescence (generally coupled with a carrier protein), quantification of the injection volume by means of the detection system. When using our Femtotips, the determined settings can be retained from capillary to capillary. Typical volumes are 0.1 to 0.5 pl for injection into the cytoplasm and 0.01 to 0.05 pl for injection into the nucleus. Publications: a) Ansorge, W. and Pepperkok R., (1988): Performance of an automated system for capillary microinjection into living cells. Biochem. Biophys.Meth.16, 283-292 (Calculation by injection with fluorescence markers.) b) G. Minaschek, J. Bereiter-Hahn, and G. Bertholdt (1989): Quantification of the Volume of Liquid Injected into Cells by Means of Pressure, Experimental Cell Research 183, 434-442 (Explanation of the calculation of the injection volume depending upon pressure and time.)

What can you recommend with regard to sample preparation of the injection solution for microinjection?- Samples should always be centrifuged (for 15 minutes at maximum speed in a microcentrifuge) immediately before the capillaries are loaded. - Use the Eppendorf Microloader for filling Femtotips (from the rear). Use it only once. The liquid should be extracted from the top of the tube. Make sure that no gas bubbles are in the glass capillary. If your solution contains proteins, you should work as quickly as possible after the capillary has been loaded. If the injection solution is not introduced into the medium immediately, there is a danger of the injection solution drying in the capillary, thus blocking the Femtotips. Please find more detailed information in our Applications No. 8 "Sample preparation for microinjection", which we are happy to send you upon request.

What guidelines are available for the optimal concentration of protein or plasmid in the injection capillary?As a rule, 20-200 ng/µl DNA and 1–5 mg/ml protein are injected. With an antibody solution, the concentration should be 10 to 50 times higher than that used in the in vitro test system (e.g. Southern blot).

Do you have a publication which explains how to determine the diameter of microinjection capillaries and also indicates the influence of the diameter on the injection volume?Yes, we do have one such application: Experimental Cell Research 210, 260-267 (1994): Microinjection Technique: Routine System for Characterization of Microcapillaries by Bubble Pressure Measurement,

Is any literature available on the subject of microinjection of adherent cells using Eppendorf devices?yes, please refer to our reference database, key word InjectMan NI2, available online http://www.eppendorf.de/int/index.php?&action=library&librarynode=28820&reforder=1&refcount=10&contentid=14

Can you recommend any parameters for the injection of DNA into adherent cells?Parameters for the injection of DNA into mammalian cells using Femtotip or Femtotip II: pc = 30 to 300 hPa pi = 50 to 500 hPa ti = 0.3 to 1.5 s The parameters must be optimized according to each experiment. If you are using Femtotips, please mount the universal capillary holder at an angle of 45 °.

What is the typical injection volume?The injection volume is typically ~ 10 % of the cell volume.

Can you supply us with literature about microinjection of DNA or proteins into adherent cells?Our Application No. 8 contains interesting hints about the preparation of samples for microinjection. www.eppendorf.com

Which dextran is most suitable for microinjection?All dextrans can be used irrespective of the molecular weight. For a special application, namely compartment-specific injections, we use dextran with a molecular weight in excess of 60 kDA, as it cannot penetrate the core barrel.

Which Eppendorf devices do you recommend for the injection into insect embryos?We recommend TransferMan NK 2 or InjectMan NI 2, FemtoJet express and a footswitch for the FemtoJet express. For objects with a strong shell (Chitin) a Piezo Drill may be necessary.

Which injection pressure do I have to select for microinjection into fish embryos?Microinjections into embryos are normally performed manually with an injection pressure of 300-500 hPa and a very short injection time of 0.1-0.2 seconds. The high injection pressure prevents the injection needle from clogging.

What is the typical injection volume when injecting into drosophila embryos?The typical injection volume ranges between 60-100pl

Can you supply us with literature about microinjection into C. elegans?Yes, our Userguide No 0112/06 “Microinjection of plasmid DNA or double stranded RNA into the gonads of C. elegans” contains lots of useful information about this kind of application. The Userguide is available at our website

Can you recommend any literature about microinjection into plant cells?1. Schnorf, M. et al., An improved approach for transformation of plant cells by microinjection: molecular and genetic analysis, Transgenic Research 1, 23-30 (1991) 2. Brücker et al., Microinjection of heme oxygenase genes rescues phytochrome-chromophore-deficient mutants of the moss Ceratodon purpureus, Planta 210, 529-535 (2000)

What is important for the microinjection into plant cells?For the injection into plant cells, a pressure greater than 7,000 hPa is very often necessary. Therefore, a CellTram Oil or vario should be used for plants with a high turgor (max. pressure 20.000 hPa).

What equipment is required to inject fluorescent dyes into plant cells and to then aspirate the entire content of the cell?Since a very high pressure is required for injection into some plant cells, we recommend using the manual CellTram Oil Microinjector in combination with a micromanipulator (e.g. TransferMan NK 2), which enables material to be extracted out of the cell. If suspension cells are used, a second micromanipulator is necessary to fix the cells.

What should be taken into account when injecting proteins? .It is sometimes difficult to inject protein solutions, as any contamination can block the capillary. Therefore, the solutions must be prepared very carefully (e.g. cleaned by centrifugation columns or ultracentrifugation). Before loading the Femtotip, the solution should always be centrifuged for at least 10 minutes at the highest speed

What is the typical injection volume when injecting into drosophila embryos?The typical injection volume ranges between 60-100pl

Is it possible to control the manipulators and elctronical injectors via PC?Yes, you ca nfind a detailled description on our homepage

Which grip head is included in the delivery package of the FemtoJet/FemtoJet express?Grip head number 0. It can be used for capillaries with an outer diameter of 1,0-1,1 mm.

What is the outer diameter of the universal capillary holder?The outer diameter is 4 mm.

I want to use my own capillaries with Eppendorf microinjectors. Which capillary grip would fit my tips?All Eppendorf microinjectors (Transjector, FemtoJet/FemtoJet express and CellTram) are equipped with the Universal Capillary Holder. Different grip heads can be interchanged for the use of capillaries with different outer diameters. Use the table below to determine which grip head suits your needs. Description Capillary grip 0: fits microcapillaries with an outer diameter of 1.0 to 1.1 mm, order no. 5176 210.003 Capillary grip 1: fits microcapillaries with an outer diameter of 1.2 to 1.3 mm, order no. 5176 212.006 Capillary grip 2: fits microcapillaries with an outer diameter of 1.4 to 1.5 mm, order no. 5176 214.009 Capillary grip 3: fits microcapillaries with an outer diameter of 0.7 to 0.9 mm, order no. 5176 207.002

How can the different grip heads be distinguished?The grip heads have different numbers of notches. The grip head 0 has no notch at all; the grip head 3 has, for example, three notches

Can a PC be used to control the the New Manipulator Generation (NMG)?A PC can easily control all Eppendorf manipulators of the new generation. A detailed description can be found at our website.