SUBCULTURING CELLS (for subculturing one confluent flask of cells)

ERDC TN-DOER-C30

February 2003

1) Warm supplemented MEM, trypsin-EDTA, and PBS in 37 C water bath. Place

an aliquot each of Hygromycin B and Zeocin in the alcohol bath.

2) Siphon off old medium from the T-75 flask. Transfer 10 mL of PBS into the flask.

Wait for about 2 min before pipetting off the PBS.

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

for 5-10 min.

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

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

CAUTION: These cells should not stay in trypsin for more than 15 min.

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

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

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

cells (see "COUNTING CELLS").

8) Centrifuge the 13 mL of cell suspension at 1,000 rpm's for 5 min.

9) 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.

10) If the cells are not used for an assay, transfer the required volume of cell sus-

pension 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.

11) Pipet 30 L each of 100-mg/mL Hygromycin B and Zeocin solution into the flask.

12) 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.

3) Count cell numbers in 5 sections on both sides of the hemacytometer. The total

number of sections counted should be 10.

4) Add up the number of live and of dead cells for all 10 sections.

5) Multiply the total number of live cells by a factor of 104 to obtain the number of

live cells/mL in the cell suspension. Likewise, the dead cells/mL is obtained by

multiplying by the same 104 factor. The viability of the cell suspension is the

total number of live cells divided by the total (live and dead) number of cells.