Fetal Bovine Serum (FBS), stored frozen

ERDC TN-DOER-C10

March 2000

c) Connect tubings on the filter to the aspirator and to the beaker containing

ultrapure water.

d) Turn on the aspirator.

e) When the bottle is filled, turn off the aspirator and disconnect the tubings.

This filtered water can be stored in the refrigerator until further use.

3) Fetal Bovine Serum (FBS), stored frozen.

a) Remove FBS from freezer and place in 37 C water bath.

b) When thawed, wipe the outside of the container with a dry paper towel

followed with a paper towel moistened with 70 percent alcohol.

c) Aseptically transfer 50-mL aliquots of FBS into sterile 50-mL centrifuge tubes.

d) Cap tubes and store in -20 C freezer until further use.

4) Trypsin, 1x, stored frozen.

a) Remove 1x trypsin stock (100 mL) from -20 C freezer and place in 37 C

water bath.

b) When thawed, wipe the outside of the container with a dry paper towel

followed with a paper towel moistened with 70 percent alcohol.

c) Aseptically transfer 6-mL aliquots of 1x trypsin into 15-mL sterile centrifuge

tubes.

d) Cap tubes and store in -20 C freezer.

5) Geneticin (G418), 40 mg/mL or 100x solution, stored frozen.

a) Dissolve 1 g of Geneticin (G418) disulfate salt in 25 mL of sterile-filtered,

ultrapure water.

b) Transfer 1.5-mL aliquots in sterile cryovials.

c) Store in the -20 C freezer.

d) NOTE: The Geneticin concentration needed to maintain selection pressure

on these recombinant cells is 0.4 mg/mL. In a T-75 flask containing 15 mL

of medium, add 150 l (or 10 percent of medium volume) of the 40 mg/mL

Geneticin stock. Geneticin is added to the medium in every cell passage.

6) Trypan Blue, 0.05 percent, stored refrigerated, nonsterile.

a) Transfer 6.3 mL of 0.4 percent Trypan Blue solution (stored at room

temperature) into 43.7 mL of PBS solution in a beaker and mix well.

b) Pipet 1.800 mL of the 0.05 percent Trypan Blue solution with a micropipettor

into each culture tube. The solution prepared provides 27 aliquots.

c) CAUTION: Trypan Blue is a suspected mutagen. Wear gloves at all times

when handling this reagent.

d) Cover the neck of each culture tube with a piece of parafilm.