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Efficient DNA transfection of primary CNS neurons using TransMessenger ,,, Transfection Reagent

after transfection of a GFP-encoding plasmid; C: fluorescence image of live cells 24 hours after transfection (same visual field as B). Cells were transfected in a 96-well format using 0.3 g of DNA and 2 l of TransMessenger Reagent per well.

To distinguish the cell types in the CryoCell cultures, cell-specific markers were detected using monoclonal primary antibodies (anti-MAP2 antibodies for neurons, anti-GFAP antibodies for astrocytes, the major subtype of glial cells in the brain). "Excellent Morphology of CryoCell Cultures" shows a typical result. Staining with anti-MAP2 antibodies reveals a well-developed neurite network and brightly stained somata, indicating good morphology and differentiation of the neurons. Staining with anti-GFAP antibodies shows glial-cell astrocytes with a stellate morphology. Serum-free cultures contain many fewer astrocytes than serum-containing cultures (data not shown). Transfected and untransfected cultures display the same GFAP- and MAP2-staining pattern. The immunofluorescence results show that primary cultures of cortical neurons generated from CryoCells have morphologies and characteristics comparable to cultures derived from freshly prepared cells.

Excellent Morphology of CryoCell Cultures Cells stained with anti-MAP2 primary antibodies and Cy3-labeled secondary antibodies, 24 hours after transfection: A: Untransfected cells; B: transfected cells, anti-MAP2 staining; C: transfected cells, GFP fluorescence; D: superimposed images from B and C. Cells stained with anti-GFAP primary antibodies and Cy3-labeled secondary antibodies: E: Untransfected cells; F: transfected cells, anti-GFAP s
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