SUBCULTURING CELLS (for subculturing one confluent flask of cells)

ERDC TN-DOER-C10

March 2000

12) Add 200 L of 40 mg/mL Geneticin solution into the flask.

13) Place flask in the CO2 incubator to allow the cells to attach overnight.

14) Siphon off the medium and transfer 15 mL of fresh medium and 150 L of

Geneticin the next day. Incubate cells.

15) Check cell growth daily. A successful thawing of frozen permanents will show

confluent growth of cells within a week.

SUBCULTURING CELLS (for subculturing one confluent flask of cells).

1) Warm supplemented MEM, trypsin, and versene (EDTA) in 37 C water bath.

Thaw out a cryovial of Geneticin in the alcohol bath.

2) Siphon off old medium from the T-75 flask. Transfer 2 mL of versene into the

flask; this volume should just cover the surface of cells. Wait for about

2 minutes before pipetting off the versene.

3) Add 3 mL of freshly warmed trypsin and place the flask in the incubator for

5-10 minutes.

4) Tap sides of the flask sharply to loosen cells (incubate the flask for another

3-5 minutes if the cells are not detached from the flask).

5) CAUTION: These cells should not stay in trypsin for more than 15 minutes.

6) Add 10 mL of MEM. Triturate cell suspension to break up any cell clumps.

7) Place the cell suspension into a sterile 15-mL centrifuge tube.

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

cells (see following section, "COUNTING CELLS").

9) Centrifuge the 13 mL of cell suspension at 1,000 rpm for 5 minutes.

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

pellet. Resuspend cell pellet in 10 mL of fresh medium, or any volume

appropriate for an assay.

11) If the cells are not used for an assay, transfer the required volume of cell suspension

based on the number of cells counted into a new T-75 flask. Seed about 2 to 3 106

cells per T-75 flask (typically, a flask seeded with 3 106 cells will be confluent in

4 to 5 days). Add fresh medium to give a total of 15 mL.

12) Pipet 150 L of 40 mg/mL Geneticin solution into the flask.

13) Incubate flask.

COUNTING CELLS.

1) Transfer 200 L of cell suspension that has been triturated into the test tube

containing 1.800 mL of freshly warmed 0.05 percent Trypan Blue solution.

2) Mix the suspension thoroughly and apply about 10 L to each side of the

hemacytometer under the cover glass.