( sing 200 ng of DNA in combination ...)Effectene Transfection Reagent provides efficient gene delivery ,,, to primary neuronal cell cultures

sing 200 ng of DNA in combination with 25 l of Effectene Reagent per well. For culture and transfection conditions, see Materials and methods.

Briefly, fixed and permeabilized cells were incubated for 1 hour at room temperature with a monoclonal anti-microtubule-associated protein 2 (MAP2) antibody. Cells were washed and incubated with a Cy3-conjugated secondary antibody for 45 minutes at room temperature.

Results and discussion

Figure 1 shows transfection efficiency in randomly chosen view-fields, as determined by counting the number of cell nuclei, MAP2-positive neurons, and GFP-positive fluorescent neurons. The highest transfection efficiency was obtained using 200 ng of DNA with 25 l of Effectene Reagent. Under these conditions, Effectene Reagent is also suitable for transfection of glial cells (data not shown). Use of 10 l Effectene Reagent or exposure of cells to complexes for longer than 5 hours was toxic to neurons. Figure 2 shows two MAP2-positive transfected neurons (green) in a culture that was treated with 200 ng DNA and 5 l of Effectene Reagent. Nuclei (blue) of cells not reacting with MAP2 antibodies are glial-cell nuclei. Note that the nuclei of transfected neurons are not fragmented, which is an indicator of good cell health. In control transfection experiments (i.e., when the plasmid lacking the eukaryotic promoter was used), green fluorescence was not observed.

Transfected Primary Neuronal Cells

Figure 2 Fluorescence micrographs of transfected primary cultures from rat superior colliculus. On day 9 in vitro, cultures were treated with 200 ng of EGFP plasmid DNA complexed with 5 l of Effectene Reagent. Cells w
'"/>

Source:


Page: All 1 2 3 4
Related biology technology :

1. Effectene Transfection Reagent
2. Highest Transfection Efficiency of an Endotoxin-Sensitive Mammalian Cell Line
3. Optimizing Transfection Conditions for Studying Signal Transduction Pathways
4. Transfection of Green Fluorescent Protein into Human Adrenalcarcinoma Cells
5. Improve Lipid- or Calcium Phosphate-Mediated Transfection of Human Dermal Fibroblasts
6. Versatile Transfection Reagent Offers Low Toxicity and Consistent Performance
7. Low-Toxicity, Lipid-Mediated Transfection of Mammalian Cells
8. Mycoplasma Contamination Reduces the Effect of Lipid-Mediated Transfection of Mammalian Cells
9. High-Efficiency Transfections Achieved with New Low-Toxicity Reagent
10. Efficient Transfection of Neurospora Crassa
11. Eppendorf Multiporator Transfection Protocols for Eukaryotic Cells