Diolistic labeling of neuronal cultures and intact tissue using a hand-held gene gun.
Diolistic labeling is a highly efficient method for introducing dyes into cells using biolistic techniques. The use of lipophilic carbocyanine dyes, combined with particle-mediated biolistic delivery using a hand-held gene gun, allows non-toxic labeling of multiple cells in both living and fixed tissue. The technique is rapid (labeled cells can be visualized in minutes) and technically undemanding. Here, we provide a detailed protocol for diolistic labeling of cultured human embryonic kidney 293 cells and whole brain using a hand-held gene gun. There are four major steps: (i) coating gold microcarriers with one or more dyes; (ii) transferring the microcarriers into a cartridge to make a bullet; (iii) preparation of cells or intact tissue; and (iv) firing the microcarriers into cells or tissue. The method can be readily adapted to other cell types and tissues. This protocol can be completed in less than 1 h.
O'Brien
John A
JA
Neurobiology Division, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.
Lummis
Sarah C R
SC
D019470:Biolistics / Q000379:methods*; D002232:Carbocyanines; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D006801:Humans; D009474:Neurons / Q000166:cytology*; D013194:Staining and Labeling / Q000379:methods*
D002232:Carbocyanines; C072024:carbocyanine dye DiIC12(3)