Amaxa transfection

Keywords Migration, Chemotaxis, Neutrophil, HL-60, Actin cytoskeleton, Amaxa transfection. Micropipette, EZ-TAXIS assay... 

As an alternative to the chemical transfection systems, the primary cells can be effectively transfected by electroporation (Appendix 5). Amaxa has developed a nucleofection (electroporation) system that is very effective for delivering expression vectors into cells at high efficiency and low cellular mortality. The step-by-step conditions for the transfection of numerous primary cell types, including human airway epithelial cells, can be found at http //www.amaxa.com/primary-cells.html. 

Use the Amaxa Human T Cell Nucleofector Kit for unstimulated human T cells and the corresponding transfection program for human T cells following the vendor s instructions. 

S). Differentiated HL-60 cells (dHL-60) can then be used in chemotaxis assays and to visualize the cytoskeleton of a polarized cell (4). Amaxa nucleofection may be used in dHL60 cells to knock genes down (S-8) or transfect cells with a fluorescent tag to analyze protein localization (our unpublished results). Cell behavior can be analyzed either in response to a point source of chemoattractant (9-11), or using the EZ-TAXIS system, which allows simultaneous measurement of directionality and speed for six different assay conditions (12, 13). 

Fig. 2. Transient transfection of FIL-60 cells with amaxa nucleofection. Spin 5 million cells at 100 x g. Aspirate supernatant and resuspend pellet in 100 pi transfection solution per reaction and nucleofect with amaxa program Y-001. Flush with prewarmed recovery medium and incubate in an Eppendorf tube for 30 min. Transfer to a 6-well dish with...

Our protocol differs significantly from the standard Kit V protocol from amaxa, because we have optimized for cells to retain chemotactic function (not necessarily highest transfection efficiency). 

---