海马神经元细胞转染的实验技术方法相关交流-蚂蚁淘在线

ABSTRACT

This protocol describes two transfection methods for expressing GFP-tagged actin in primary neurons. The lipid reagent DOTAP (Roche Diagnostics) method produces actin-GFP-expressing hippocampal neurons that survive well during long periods in culture. The calcium phosphate method can be used to transfect neurons that have already been growing on coverslips in vitro. Transfected cells suitable for imaging can be obtained in cultures up to 15 days in vitro. One to two percent transfected cells is a typical result. A disadvantage of the calcium phosphate method is that hippocampal neurons become "fragile" after treatment.

MATERIALS

Reagents

Actin-GFP plasmid DNA (purified)
BES-buffered saline (BBS), 2X (for calcium phosphate method)
- BES, 50 mM
- Na₂HPO₄, 1.5 mM
- NaCl₂, 280 mM
CaCl₂, 250 mM (for calcium phosphate method)
DOTAP (Roche Diagnostics)
Glial monolayer culture (Spector et al. 1998a)
Glial-conditioned medium
Glucose
Hank's balanced salt solution (HBSS); Ca^⁺⁺-Mg^⁺⁺-free (also available from GIBCO) (for DOTAP method)
- NaCl, 8.0g/liter
- KCl, 0.4g/liter
- KH₂PO₄, 60 mg/liter
- Na₂HPO₄ anhydrous, 47.86 mg/liter
- glucose anhydrous, 1g/liter
- NaHCO₃, 0.35g/liter
- Sterilize and refrigerate.
Modified HEPES-buffered saline (MHBS), prewarmed (for calcium phosphate method)
- 135 mM NaCl
- 1 mM Na₂HPO₄
- 4 mM KCl
- 2 mM CaCl₂
- 20 mM HEPES buffer (Sigma)
- Make up 100 ml, adjust the pH to 7.5, and filter-sterilize.
Horse serum (for DOTAP method)
Modified Eagle's medium (MEM), (Invitrogen) (for DOTAP method)
Neurons (freshly prepared from E18 rat hippocampus for DOTAP method, or growing on coverslips for calcium phosphate method)

Equipment

Bacterial culture dish, 10-cm diameter (for DOTAP method)
Conical tube, 15-ml polystyrene (for DOTAP method)
Coverslips, nitirc acid treated and sterilized (Spector et al. 1998a)
Coverslips, with paraffin "feet" (Spector et al. 1998a)
Culture dishes, 12-well
Culture pipette
Incubator, preset to 37°C; 2.5% CO₂ (for calcium phosphate method, Step 12)
Incubator, preset to 37°C; 5% CO₂
Inverted microscope
Microcentrifuge tubes, 1.5-ml, sterile (for calcium phosphate method)
Vortex mixer
Water bath preset to 37°C (for DOTAP method)

METHOD

DOTAP Method

Resuspend neurons freshly prepared from E18 rat hippocampus in HBSS (Ca⁺⁺-Mg⁺⁺-free), and adjust to 10⁶ cells per milliliter.
In a 15-ml polystyrene conical tube, add 15 µl of DOTAP to 5 ml of MEM supplemented with 0.6% glucose (HC-MEM). Mix by vortexing. Add 1 ml of cell suspension (10⁶ cells), and mix gently by swirling the tube.
Incubate the tube for 15 minutes in a 37°C water bath. Add 3 µg of purified plasmid DNA diluted in 100 µl of HBSS to the cells, and mix gently. Incubate for 30-40 minutes at 37°C.
Prepare 15 coverslips with paraffin "feet" and place in a 10-cm-diameter bacterial culture dish. Gently resuspend the cells. Plate them in HC-MEM with 10% horse serum onto the prepared coverslips.The density of the culture can be adjusted by using between 2 x 10⁵ and 10⁶ cells per dish. Draw the cells gently up and down in the culture pipette immediately before plating to ensure they are evenly distributed over the dish.
Incubate for 2-3 hours at 37°C. Transfer the coverslips with the attached neurons to 12-well culture dishes containing pre-prepared glial monolayers. "Flip" the coverslips so that the neurons are underneath, facing the glia. Return the culture dishes to the incubator immediately, as the pH of this medium is not stable for long outside the incubator.This method should yield 50 or more actin-GFP-expressing cells per 18-mm coverslip. Calcium Phosphate Method
Prepare a 12-well culture plate with 2 ml of glial-conditioned medium in each well. Equilibrate by placing it in an incubator with an atmosphere of 5% CO₂ for at least 30 minutes at 37°C.The correct pH is crucial to obtaining the fine CaPO₄ precipitate necessary for optimum transfection efficiency. Check that the phenol red indicator in the medium is pink before using it. If the medium is too alkaline (phenol red indicator is red), the precipitate will form too quickly, whereas if the medium is too acid (phenol red indicator is yellow), the precipitate will not form or will form too slowly.
Transfer the coverslips with the neurons from the original well in which they have been growing to the equilibrated glial-conditioned medium in the new plate. If using the Banker culture method, turn the coverslips over so that the neurons are on top.
Store the original plate with the neuronal culture medium in the incubator for reuse after the transfection procedure.
Perform Steps 9-11 rapidly. For each coverslip, prepare a sterile 1.5-ml microcentrifuge tube with 5 µg of actin-GFP plasmid DNA in 60 µl of 250 mM CaCl₂.
Add 60 µl of 2X BBS to the DNA. Vortex several times during the addition.
Immediately add the DNA to the cells (120 µl/well) and gently mix by swirling the plate.
Incubate the cells in this transfection medium for 30-90 minutes at 37°C and 2.5% CO₂. After 30 minutes, begin checking for precipitate formation using an inverted microscope. It should appear as fine black speckles on the coverslip. If precipitate has not formed, continue the incubation and reexamine at 30-minute intervals.
As soon as the precipitate has formed, gently wash the coverslips twice with prewarmed MHBS (1 ml/wash). Transfer them to the original plate from Step 8 that has been stored at 37°C and 5% CO₂, and return the cells to standard culture conditions. Good results are highly dependent on the quality of the calcium-phosphate precipitate, whose production requires some practice. Effectene (QIAGEN), used as an alternative to calcium phosphate, is technically easier, but results with hippocampal neurons are generally inferior to CaPO₄. A recently introduced electroporation device, the Nucleofector, produced by Amaxa (http://www.amaxa.com), gives superior results compared to previous transfection techniques, yielding ~30% GFP-actin-expressing cells for rat hippocampal neurons.

DISCUSSION

Two methods for low-density culture of rat hippocampal neurons are widely used. The original method developed by Bartlett and Banker (1984a) yields excellent results. An advantage for high-resolution imaging is that the neurons are maintained on coverslips independently of the supporting astroglial cells, which are attached to the culture dish. In a simpler and faster alternative, neurons are grown in a serum-free chemically defined medium, Neurobasal/B27 (Invitrogen-GIBCO), without astroglial feeder layers. A disadvantage of this medium is that hippocampal neurons develop "twisted" dendrites and tend to retain filopodia-like dendritic spines of immature appearance even after more than 3 weeks in culture. In contrast, the same cells grown in "Banker" cultures for 3 weeks are virtually free of filopodia and have dendritic spines equivalent in appearance to those of mature brain (see Bartlett and Banker 1984b). These same considerations regarding culture conditions also apply to low-density cultures of hippocampal neurons established from transgenic mice. Successful high-resolution imaging of the neuronal cytoskeleton requires a precision-machined observation chamber, a suitable medium for maintaining neurons in good condition during long periods of viewing (minutes to hours), and careful control of temperature, which is essential for maintaining stable focus. Precision imaging chambers developed for this purpose are available from Life Imaging Services (http://www.lis.ch/thechamber.html). The best means of maintaining effective temperature control is to mount the microscope together with ancillary equipment in a warm-air environment box such as "The Cube" from Life Imaging Services.

REFERENCES

Bartlett, W.P and Banker, G.A. 1984a. An electron microscopic study of the development of axons and dendrites by hippocampal neurons in culture. I. Cells which develop without intercellular contacts. J. Neurosci 4: 1944-1953.[Abstract] Bartlett, W.P. and Banker, G.A. 1984b. An electron microscopic study of the development of axons and dendrites by hippocampal neurons in culture. II. Synaptic relationships. J. Neurosci. 4: 1954-1965.[Abstract] Spector, D.L., Goldman, R.D., and Leinwand, L.A. 1998a. Cells: A laboratory manual, Vol. 1, pp. 9.1-9.13. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Anyone using the procedures in this protocol does so at their own risk. Cold Spring Harbor Laboratory makes no representations or warranties with respect to the material set forth in this protocol and has no liability in connection with the use of these materials. Materials used in this protocol may be considered hazardous and should be used with caution. For a full listing of cautions regarding these material, please consult: Live Cell Imaging: A Laboratory Manual, by Robert Goldman and David Spector, © 2005 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, p. 513-521.

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