BIO RAD Helios Gene Gun System, Инструкция по эксплуатации

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Section 6 Preparation of Mammalian Cell Targets
32 | Helios Gene Gun System
Section 6
Preparation of Mammalian Cell Targets
Time Considerations: Setting up the device requires no more than 5 min, while the delivery process
requires approximately 30–60 sec per cell target. If necessary, sterilize parts as described in Cleaning and
Sterilizing the Helios Gene Gun, in Section 10, before starting.
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 Particle Delivery Using the Helios Gene Gun,
in Section 5.
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 spacer 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.