Bio-Rad PDS-1000 / He™ and Hepta™ Systems - Manual (Page 27)

4. Coating microcarriers with DNA
The day of the scheduled bombardment, coat the microcarriers with DNA. To obtain the
best results, use the DNA-coated microcarriers as soon as possible.
5. Loading DNA-coated microcarriers onto a macrocarrier/macrocarrier holder
Each macrocarrier is placed inside a macrocarrier holder and sterilized, as described
above. Prior to the application of the DNA-coated microcarriers onto a macrocarrier, pre-
pare a small desiccating chamber for each macrocarrier/macrocarrier holder and place
away from excessive vibration.
A small desiccating chamber consists of a sterile 60 mm tissue culture Petri dish (with lid)
containing CaCl
2
as desiccant in the base of the dish (Figures 4.5 and 4.6). The desiccant is
covered with a small piece of filter paper to provide a stable platform for the macrocarrier/
macrocarrier holder. The sterile macrocarrier/macrocarrier holder is placed atop the filter
paper, with the macrocarrier facing up and the stainless steel holder touching the filter paper.
Fig. 4.5A and 4.5B. Loading DNA-coated microcarriers onto a macrocarrier/macrocarrier holder,
positioned in small desiccating chamber.
This environment permits rapid access to each macrocarrier and provides a low humidi-
ty environment for the ethanol to uniformly evaporate from the microcarriers. This low humid-
ity, along with a minimum of vibration during evaporation, minimizes microcarrier
agglomeration.
For each macrocarrier, remove 6 µl aliquots (approximately 500 µg) of microcarriers and
spread evenly over the central 1 cm of the macrocarrier using a pipette tip. Pipet from a con-
tinuously vortexed tube and rapidly apply suspended microcarriers to the macrocarrier, as
microcarriers quickly settle out from the ethanol solution in the tube or even in the pipette tip.
Immediately cover the culture dish after application of the microcarrier suspension to the
macrocarrier. The ethanol should evaporate within 10 minutes to leave the DNA-coated micro-
carriers adhering to the macrocarrier. The loaded macrocarriers should be used within 2 hours.
6. Cleaning chamber walls
Clean the chamber of the PDS-1000/He as desired with 70% ethanol. Allow time for dry-
ing. Do not autoclave or flame sterilize the PDS-1000/He unit.
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A B