( have adhered securely to the ...)Biolistic Transfection of Organotypic Brain Slices

Coating Microprojectiles with DNA
1.6 m gold particles are prepared using the guidelines for coating microprojectiles provided in the Bio-Rad instruction manual, except for two modifications. First, in the agglomeration step, we vortex the gold for a longer period of time to reduce clumping( 4 minutes for each wash instead of 2 minutes as recommended). Second, after all of the reagents are added to the gold in the DNA precipitation step, we vortex for 2 minutes, then intermittently for the next 20 minutes. The additional time in this step allows for improved coating of the gold particles.

Coating Macrocarrier Disks with Gold
Transfection of dissociated cells requires only a few simple modifications to the standard Biolistic bombardment protocol. Because the percentage of successfully transfected cells varies with the density of gold particles as well as with cell density, we typically use 1.5 times the standard microprojectile recipe to coat six macrocarrier disks (i.e. 3 mg gold, 5 g plasmid/macrocarrier). At greater than two or three times the standard density, however, cell death becomes significant. For co-transfections of two different expression constructs, one-half of the standard amount of each DNA construct is used, such that the total amount of DNA used per recipe remains the same.

Transfecting Cell Lines
To transfect dissociated cells, the media is removed from the culture dish as thoroughly as possible without completely drying the cells; aspirating media into a sterile serological pipette works well. Blasting should proceed quickly from this point, to prevent dessication of the cells. The aspirate
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