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We use CP for transfecting exogenous proteins into cultured hippocampal neurons and subsequent localization studies using immunofluorescence or video microscopy. CP can also be used for small-scale biochemical studies like western blot to characterize an antibody or to verify the fulllength expression of a transfected protein.
warmed to room temperature
Na2HPO4, 50 mM BES [N,N-bis(2- hydroxyethyl)-2-aminoethanesulfonic acid]; Sigma, St. Louis, MO, USA), pH 7.1
[4-(2-hydroxyethyl)-1-piperazineethanesufonic acid], pH 7.1, 4 mM KCl, 1 mM Na2HPO4, 2 mM CaCl2, 2 mM MgCl2, 10 mM glucose)
For optimal results, prepare endotoxin- free DNA with a plasmid purification kit (EndoFree Plasmid Maxi Kit; Qiagen, Hilden, Germany). After the final precipitation, dissolve DNA in endotoxin-free
water or Tris-EDTA (TE) at a concentration of 1 to 3 mg/mL. Prepare 50 to 100-μL aliquots and store at -20°C. Store thawed DNA at 4°C and avoid frequent freezing and thawing. Preparation of Conditioned N2 Older, polarized neurons survive better in medium enriched in factors secreted by astrocytes. To keep neurons in conditioned medium throughout the transfection, it is necessary to have preconditioned medium at hand. Therefore, we grow astrocytes on 6-cm plastic dishes as described in Reference 2. The N2 medium in which astrocytes are grown can be used for transfection. Check that the pH is not too acidic, as this will prevent the precipitate to form.
1. Warm 2× BBS and CaCl2 to room temperature. Place HBS in a 37°C waterbath. 2. Add 2 mL of conditioned N2 into a 3.5-cm dish and transfer coverslips into the dishes such that the neurons are facing up. Immediately place them in the 5% incubator for 30 minutes. 3. In a 2-mL tube mix 2 to 5 μg of DNA with the CaCl2 solution to a final volume of 60 μL. Mix briefly by vortex mixing. 4. Slowly add 60 μL of 2× BBS while stirring with the tip. Vortex mix briefly. If several dishes are transfected with the same DNA, prepare a master mixture. This ensures equal transfection for all dishes. 5. After 1 minute, take the cells out of the incubator and check the pH of the medium. If necessary, add drops of diluted hydrochloric acid to reach the desired pH. Color must be red, not yellowish and not purple. 6. Add DNA–phosphate precipitate and stir gently. The medium should now become yellowish. Transfer dishes to a 2.5% CO2 incubator. 7. Let precipitate form and settle onto cells for 30 to 120 minutes. Check the medium after 30 to 60 min. If the medium is very purple and turbid, the precipitate is probably already very big. Check on a microscope for size of the precipitate. Precipitate should have the size and appearance of bacterial contamination. Big precipitates are toxic, and the cells will die. 8. After precipitate has formed, gently wash the cells 2 times with prewarmed HBS, then add the original medium in which they were grown. 9. Flip coverslips so that the neurons are facing the bottom of the dish and incubate until analysis. Depending on the construct, we analyze cells 8 hours to 3 days after transfection.
Several parameters are critical for an efficient transfection. 1. We found that the use of endotoxinfree DNA significantly increases the number of transfected cells. 2. The use of a 2.5% CO2 incubator during the transfection procedure is essen tial in our hands. 3. Successful transfection is dependent on the pH of the medium to which the precipitate is added. A too acidic medium will prevent the precipitate to form, and a too alkaline medium will lead to huge precipitates that are neurotoxic.
Even by carefully following the protocol transfection, efficiencies vary from transfection to transfection. Moreover different constructs generally give different efficiencies, even if the same expression vector is used (C.K. and F.R., unpublished observation). Also, transfected cells on a dish or coverslip are not evenly distributed, but tend to cluster.
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