Optimizing microinjection parameters ●TIMING 3-5 min
1| To set up compensation pressure, load 1 μl of 0.1% Fast Green into injection needle with a microloader.
▲ CRITICAL STEP Avoid any air bubble inside of the glass capillary after loading.
2| Place a 35 mm petri dish filled with appropriate culture medium on the platform of microscope.
3| Fix the injection needle to the holder of micromanipulator and position the needle tip in the medium.
4| Observe the discharge of visible Fast Green from the needle tip. Adjust compensation pressure (Pc) from the control panel of FemtoJet to a value which ensures a continuous trace amount of discharge around of the needle tip.
▲ CRITICAL STEP If the compensation pressure is too high, the constitutive discharge from the injection needle may cause high background of marker dye DTR. If the compensation pressure is not enough, the injected substance may never get pushed out of the needle tip.
5| To set up injection pressure, adjust injection pressure (Pi) from the control panel of FemtoJet to a value double the compensation pressure Pc. For example, if Pc=50 hpa, then set Pi=100 hpa.
6| Set up injection time (Ti) from the control panel of FemtoJet according to the values listed in Table 1.
7| To determine injection volume of each injection, continually press “injection” button until 1 μl of Fast Green completely finished. The number of injection is automatically counted by the microinjector. Calculate injection volume=1 μl/number of injection.
▲ CRITICAL STEP Determination of injection volume of each injection under certain injection parameters is crucial to define amount of delivery in further experiments (see injection volumes of the common parameter settings in Table 1).
Preliminary examination before microinjection experiments ●TIMING varies from 2 h to 20 d, 1-2 days before microinjection experiments
8| Carefully take out a coverslip with cultured cells, place it into injection chamber.
! CAUTION Cell culture procedures involving animal or human tissue are to be performed in accordance with relevant authorities’ guidelines and regulations.
9| Place the petri dish onto the platform of the microscope. Adjust focusing until the structure of cytosolic and nuclear areas appear.
10| Select an area containing morphologically healthy and well-connected cells. It is preferable to inject cells in one area, which facilitates detection of the injected cells.
11| Position the needle on the top of the cell using the micromanipulator and joystick. Use “coarse” movement option to position needle into the observation field, 30-45 degree to the bottom of injection chamber, and then switch to “fine” movement option to place the needle just on the top of the cell surface, without piercing the cell membrane. Carefully lower the tip down by slowly moving the vertical control of the joystick to let the tip penetrate into the cytosolic area of the cell.
▲ CRITICAL STEP This step is a key for a successful injection and high cell survival rate after injection. Great patience and effort are appreciated for beginners to achieve successful and consistent injection results.
12| The concentration of marker dye DTR needs to be optimized before experiments. Inject various doses of DTR ranging from 10 to 1000 μg/ml into cells. Inject 100 cells in one coverslip. Fix cells with 4% paraformaldehyde and 4% sucrose for 20 min at room temperature. Wash coverslips with PBS for 5 min. Mount coverslips on a glass slide with Clarion mounting medium and observe under a fluroscence microscope.
13| Count the number of DTR positive cells that remain after injection. For example, in human and rat primary neurons, injection of DTR at 100-300 μg/ml results in the maximal detection of positive cells. A dose of 1000 µg/ml is to be toxic. Therefore, we use DTR at a final concentration of 100-200 μg/ml for our experiments with primary cells.
▲ CRITICAL STEP Since the injected substance gets diluted in dividing cells, we increase this concentration to 400 µg/ml in dividing cell lines.
14| Cell viability after injection needs to be determined. Inject 100 cells per coverslip with 200 μg/ml DTR. Fix cells after various time of injection ranging from 0 to 20 days with 4% paraformaldehyde and 4% sucrose for 20 min at room temperature. Wash coverslips with PBS for 5 min. Permeabilize cells with 0.1% Triton X-100 and 0.1% sodium citrate for 2 min on ice. Rinse coverslips twice by PBS. The label cell death using TUNEL kit as described by manufacturer. Mount coverslips and observe under a fluroscence microscope. Calculate cell death=(number of TUNEL positive cells/number of DTR positive cells)*100%.
15| The cytotoxicity of protein/peptide/cDNA active buffer needs to be examined. For example, we have studied the implication of active caspase-6 in human primary neurons. Active caspases are dissolved in an active caspase buffer (20 mM piperazine-N,N’-bis-(2-ethanesulfonic acid), 100 mM NaCl, 10 mM dithiothreitol, 1 mM EDTA, 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonic acid, 10% sucrose, pH7.2) which contains a number of chemicals that could be toxic to neurons 7. Therefore, the toxicity of the active caspase buffer in the presence of the pre-determined optimal concentration of DTR in neurons is examined. We compared cell death results of the active caspase buffer with PBS to ensure the buffer itself does not induce significant cell death.
▲ CRITICAL STEP The controls for buffers, solvents, vehicles and vectors are necessary to achieve reliable results in microinjection experiments.
According to the experiment subjects, microinjection can be performed in either attached cells (A), including primary cells and dividing cell lines, or suspended cells (B), such as oocytes, eggs and protozoan.
(A) Injecting attached cells ●TIMING 5 s per cell, 8-10 min per coverslip
Primary cells are cultured on poly-lysine coated glass or plastic coverslips to form appropriate morphology and networks. Microinject 100 cells per coverslip as described in Step 8-11 with pre-determined injection parameters (Step 1-7). Apply drug treatments after the injection if desired. The injected cells can then be returned to 37 oC incubator for desired time to allow injected substance to function or express. After incubation, cells can be fixed or collected for further analysis. Cell lines are injected similar to primary cells with higher concentrations of DTR as well as substance of interest to avoid dilution during cell division.
! CAUTION Prolonged incubation after injection is not recommended in cell lines due to the diluting effects.
(B) Injecting suspended cells ●TIMING 10 s per cell
Transfer cells into injection chamber in the appropriate medium. Fix one holding needle to the holding channel of microinjector (Fig. 3d). First position the holding needle within optical field with “coarse” movement option and then switch to “fine” movement option. Slightly turn the handset of CellTram Air to manually apply a negative pressure to suck a single cell at the tip of holding needle. With injection needle, inject substance of interest into the desired areas of cells as described in 16A. Release cell by turn the CellTram Air handset reversely to produce a positive pressure through the holding needle.
▲ CRITICAL STEP If the negative pressure is too high, the cell can be sucked into the holding pipet. Hold only one cell for a single injection.