Bio-Rad Helios® Gene Gun System - Manual (Page 33)

2. Turn the regulator valve counterclockwise until both the high and low pressure gauges on
the helium regulator register 0 psi. Several increase/decrease adjustments on the regulator
may be necessary. Listen for venting to ensure complete depressurization. The system is now
depressurized and can be safely disassembled.
3. Disconnect the helium hose from the regulator by pulling the sleeve on the Swagelok
Quick-Connect coupling toward the helium hose and pulling the fittings apart.
4. Disconnect the helium hose from the Gene Gun by pulling the sleeve on the Swagelok
Quick-Connect coupling toward the Gene Gun and pulling the fitting apart. (Warning:
For safety, do not leave the Helios Gene Gun unattended while attached to the helium
regulator.)
Section 6
Preparation of Mammalian Cell Targets
Time Considerations: Setting up the device requires no more than 5 min, while the deliv-
ery process requires approximately 30–60 sec per cell target. If necessary, sterilize parts as
described in Section 10.5 before starting.
6.1 In vitro Delivery to Adherent Cells
Method 1
Day 1
1. Trypsinize cells from flasks. Resuspend the cells in tissue culture media at a density so that
inoculating 2 ml into a 35 mm tissue culture plate will produce a monolayer 60–80%
confluent after 24 hr.
2. Inoculate 2 ml of cells into 35 mm tissue culture plates.
3. Incubate overnight under the appropriate conditions.
Day 2
1. Prepare the Helios Gene Gun for operation as described in Section 5.3.
2. Immediately prior to DNA delivery, aspirate the media from the dish.
3. 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).
4. Add 1.5–2 ml of media to the plate and return to the incubator.
Method 2
1. Trypsinize cells from flasks. Resuspend the cells in tissue culture media buffered with
25 mM HEPES (pH 7.3) and count viable cells.
2. Dilute cells to 2 x 10 6 cells/ml.
3. Using a sterile pipet tip, spread 100 µl of the cell suspension in a 1.5 cm diameter circle
in the center of a 35 mm dish, being careful not to break the meniscus. It is useful to draw
a 1.5 cm diameter circle on an index card to use as a template under the culture dish.
4. Allow the plates to incubate undisturbed for 30 min to permit the cells to attach.
5. Gently add 1 ml of media to each plate and place in the incubator. Incubate the cells for
at least 4 hr.
6. Prepare the Helios Gene Gun for operation as described in Section 5.3.
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