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 ERDC TN-DOER-C10
March 2000
Trypan Blue (0.4 percent), SIGMA #T-8154.
Dimethyl Sulfoxide (DMSO), SIGMA #D-2650.
Cell culture handling and reagent preparation throughout the assay should be carried out only
under a sterilized laminar hood using aseptic techniques. Only the last part of the P450RGS assay
(DAY 4 (ASSAY TAKEDOWN), Protocol II, "Performance of the Assay") need not be sterile.
The reagents described in the next section are prepared in bulk ahead of time and stored in
aliquots in the refrigerator or freezer. Contents and date of preparation should be properly labeled
and initialed on these reagent containers.
B. REAGENT PREPARATION.
1) Minimum Essential Medium (MEM), supplemented with L-glutamine (100x),
sodium pyruvate (100x), and FBS; stored refrigerated.
a) Remove L-Glutamine (100x), sodium pyruvate (100x), and FBS from
refrigerator or freezer and place in 37 C water bath.
b) IMPORTANT: As a precaution to avoid cell contamination, the water bath
should be cleaned and the water changed at least once a month.
c) When warmed, wipe the outside of the containers with a dry paper towel
followed with a paper towel moistened with 70 percent alcohol.
d) Under a sterile flow hood, remove 65 mL of MEM from a new bottle. This
unsupplemented medium can be saved for later use (such as in the making of
the cell freezing medium). Aseptically, transfer the following components
into the 500-mL MEM bottle:
Component
Volume, mL
Percent
MEM
435
87
FBS
50
10
L-Glutamine (100x)
10
2
Sodium pyruvate (100x)
5
1
e) Refrigerate the fully supplemented medium when not in use.
f)
IMPORTANT: Because L-Glutamine breaks down in the medium, it
should be replenished at 2 percent by volume of the remaining medium in
the bottle every 3 weeks.
2) Sterile-filtered, ultrapure water, stored refrigerated.
a) Place a 500-mL beaker of ultrapure water (not sterile) in the hood.
b) Attach the bottle-top (0.1-m VacuCap90) filter to a sterile, autoclaved,
500-mL bottle.
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