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 ERDC TN-DOER-C10
March 2000
e) Place tubes in a rack and store in refrigerator.
7) Freezing Cell Medium, made fresh each time.
a) Aseptically transfer these reagents in a sterile 15 mL centrifuge tube:
i.
3.30 mL unsupplemented MEM (66 percent).
ii.
1.25 mL FBS (25 percent).
iii.
0.40 mL DMSO (8 percent).
iv.
0.05 mL Penicillin/Streptomycin (1 percent).
b) NOTE: For a larger volume of freezing medium, these ingredients can be
increased proportionally.
c) Filter sterilize the 5 mL medium through a 0.22-m syringe filter. Keep the
filtered medium at 4 C prior to use.
III.
CELL CULTURE.
The 101L is a transgenic human cell line obtained from Columbia Analytical Services (Vista,
CA) for the P450RGS luciferase assay. These cells are shipped in cryovials frozen on dry ice and
are immediately placed in liquid nitrogen upon receipt. Whenever these cells are thawed, they are
cataloged as "passage 0." Subsamples from the original shipment have been frozen for later use.
Cells that have undergone 25 or more passages or consistently showed poor viability are terminated.
A.
STARTING THE CULTURE FROM FROZEN PERMANENTS.
1) Place supplemented MEM in 37 C water bath before thawing cells.
2) When warmed, wipe the outside of the container with a dry paper towel followed
with a paper towel moistened with 70 percent alcohol.
3) Transfer about 12 mL of MEM into a 15-mL sterile centrifuge tube.
4) Remove a cryovial of 101L cells from the liquid nitrogen storage Dewar.
5) CAUTION: Always wear a protective full-face mask, cryogenic gloves, and lab
coat during the retrieval or storage of cryovials from liquid nitrogen Dewar.
6) Thaw cryovial contents quickly (within a minute) in a 70 percent alcohol solution
bath.
7) Aseptically transfer all the thawed cell suspension into the 12 mL of medium. Mix
the cell suspension thoroughly with a sterile pipet to dilute the DMSO in the
freezing medium.
8) Centrifuge the cell suspension at 1,000 rpm for 5 minutes.
9) Siphon off the supernatant medium using a pasteur pipet attached to an aspirator
to remove any traces of DMSO.
10) Resuspend the cell pellet with 20 mL of fresh medium.
11) Transfer the cell suspension into a new T-75 flask.
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