Bio-Rad Helios® Gene Gun System - Manual (Page 48)

Discharge into Parafilm
Materials
Parafilm laboratory film (American Can Company)
Glass plate
Procedure
1. Cut a piece of Parafilm from the roll; a 1" length is required for each cartridge to be
assayed.
2. Smooth and press the Parafilm laboratory film (waxy surface down) onto the glass plate.
Fold the edges around the sides then peal away the protective paper.
3. Connect the helium regulator, helium hose, and Helios Gene Gun as described in Section 4.2.
4. Pressurize the system as described in Section 5.3.
5. Discharge a cartridge at the test psi into a strip of Parafilm laboratory film. At this point,
one can roughly determine the quality of the microcarrier preps by examining the pat-
tern of gold particles. When examining the resulting pattern, it should appear circular
with a dark center (Figure 25). The discharged cartridges should be completely empty.
Spot darkness Proportional to particle penetration
Spot width Proportional to particle spread
Spot shape Uniformity of shot
Discharge into 3% Water Agar
Materials
Slides, coverslips and mounting solution
Microscope with 10X eyepiece micrometer
3% water agar in 60 mm petri dishes
Procedure
1. Discharge a cartridge at the test psi into 3% water agar. At this point, one can roughly
determine the quality of the microcarrier preps by examining the pattern of gold parti-
cles. When examining the resulting pattern, it should appear circular with a dark center.
The discharged cartridges should be completely empty.
Additionally, one can also examine microcarrier depth penetration into the water agar
microscopically. Cut a thin slice approximately 0.5 cm long through the center of the
agar target. Mount the slice onto a slide with mounting solution and a cover slip and ana-
lyze for microcarrier depth and concentration.
2. A band of microcarriers will be visible in the agar with increasing particle sizes at
increased depths. The researcher will be able to determine areas of high, medium, and
low microcarrier density in each slice (Figure 26).
3. To determine the depth of penetration, align the 0 of an eyepiece micrometer at the upper
surface of the agar and measure the distance to the arbitrary floor of the microcarrier dis-
tribution. This value is the microcarrier depth.
The distribution pattern of the microcarriers is bell-curve shaped with respect to depth
and area. The highest microcarrier density is concentrated at the center of the shot and at the
surface of the target site. For this reason, in order to obtain a truly representative microcarri-
er distribution cross-section, be careful to examine the center and full length of your target site.
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