Bio-Rad Helios® Gene Gun System - Manual (Page 34)

7. Immediately prior to DNA delivery, aspirate the media from the dish.
8. Hold the dish perpendicular to the spacer and touch the end of the plastic spacer (if the spac-
er is sterile), or as close as possible to the target, and discharge the Gene Gun (Figure 18).
9. Add 1.5–2 ml of media to the plate and return to the incubator.
6.2 In vitro Delivery to Suspension Cultures
Method 1
1. Prepare the Helios Gene Gun for operation as described in Section 5.3.
2. Transfer the cells to a centrifuge tube and pellet in a table top centrifuge for 5 min at 250 x g.
3. Resuspend the cells in tissue culture media containing 25 mM HEPES (pH 7.3) at a con-
centration of 5 x 10 7 cells/ml.
4. Inoculate 20 µl (1 x 10 6 cells) of the cell suspension into a 1.5 cm circle in the center of
a 35 mm dish. It is useful to draw a 1.5 cm diameter circle on an index card for use as a
template under the culture dish.
5. Hold the dish perpendicular to the spacer and touch the end of the plastic spacer (if the spac-
er is sterile), or as close as possible to the target, and discharge the Gene Gun (Figure 18).
6. Add 1.5–2 ml of media to the plate and return to the incubator.
Fig. 18. Correct placement of the Gene Gun when transfecting cells in vitro (photo courtesy of
Auragen, Inc.).
Method 2
1. Prepare 35 mm tissue culture plates with Cell-Tak (Collaborative Research, Cambridge,
MA) using manufacturer’s instructions.
2. Transfer cells to a centrifuge tube and pellet in a table top centrifuge for 5 min at 250 x g.
3. Resuspend cells in tissue culture media without serum at a concentration such that inoc-
ulating 2 ml of cells into the 35 mm tissue culture plates treated with Cell-Tak will produce
a monolayer 60–80% confluent.
4. Inoculate 2 ml of cells into 35 mm tissue culture plates.
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