PROCEDURE (use sterile technique)

ERDC TN-DOER-C10

March 2000

Water bath set at 37 C.

PROCEDURE (use sterile technique).

1) Wipe down the working surfaces of the laminar flow with 70 percent alcohol.

2) Turn on the ultraviolet light and blower of the laminar flow hood. Allow 15

minutes for equilibration.

3) Turn on 37 C water bath.

4) Place supplemented MEM, versene, and one aliquot each of Trypan Blue and

trypsin in the water bath for about 30 minutes.

5) Trypsinize one confluent (T-75) flask of 101L cells with 3 mL trypsin each as

described in Section III.B, "SUBCULTURING CELLS" of Protocol I. A

confluent flask contains approximately 10 to 13 106 cells. Thus, one confluent

flask of cells should be sufficient for seeding two 96-well plates at 40,000

cells/200 L/well.

6) Transfer the cell suspension (approximately 13 mL) from the T-75 flask into a

sterile 50-mL centrifuge tube.

7) Pipet 200 L of cell suspension into the 1.800 mL of Trypan Blue solution.

Centrifuge the remaining cell suspension at 1,000 rpm for 5 min.

8) Count the cells using a hemacytometer to determine the number of cells/mL of

cultured stock (described in Section III.C, "COUNTING CELLS" of Protocol I).

9) Siphon off supernatant medium, being very careful not to remove any of the cell

pellet. Resuspend in 10 mL of fresh medium.

10) Prepare 42 mL of 200,000 cells/mL suspension from the cultured stock. Add

420 L of Geneticin to the cell suspension. This volume of cell suspension will

be sufficient for seeding two 96-well plates.

11) Example.

a) If 100 viable cells were counted in the 13-mL stock suspension, add 13 mL

of fresh medium to the cell pellet to make a 1,000,000-cells/mL suspension.

b) Then to make 42 mL of a 200,000-cells/mL suspension, transfer 8.4 mL of

the 1,000,000-cell/mL suspension into a sterile 50-mL centrifuge tube

containing 33.6 mL fresh medium.

12) Transfer the 42 mL of cell suspension into a sterile 50-mL solution basin.

13) Set the multichannel pipettor to pipet 200 L and seed the two 96-well plates.

14) IMPORTANT: To ensure that the cells are evenly distributed, mix the cell

suspension thoroughly before pipetting each plate.

15) Write the time and date on the lids of the plates.

16) Incubate plates in the CO2 incubator for 3 days.