DAY 3 (DOSING THE CELLS)

ERDC TN-DOER-C10

March 2000

III.

DAY 3 (DOSING THE CELLS).

The cells are dosed with 2,3,7,8-TCDD standards and environmental samples prepared in

iso-octane on this day. The following setup is for testing 24 environmental samples with 3 rep-

licates each.

MATERIALS AND EQUIPMENT.

2,3,7,8-TCDD stock (1 g/mL or 1,000,000 pg/mL) in iso-octane, stored in -20 C

freezer.

Environmental samples in iso-octane, stored in -20 C freezer.

Iso-octane (pesticide grade).

MEM.

Positive displacement micropipettor (10 L and 100 L).

Multichannel motorized pipettor (250 L).

Sterile pipet tips (250 L).

50-mL sterile solution basins.

1.5-mL microcentrifuge tube (1) - autoclaved.

500-L microcentrifuge tubes (8) - autoclaved.

IMPORTANT: 2,3,7,8-TCDD is highly toxic. Wear latex gloves and lab coat while handling

TCDD.

PROCEDURE.

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 in the water bath for about 30 minutes.

5) Remove TCDD standard and environmental samples (both stored in 4-mL amber

vials) from the freezer. Vortex the standards and samples and let vials warm to

room temperature.

6) Pipet off the cell medium from the two 96-well plates with a multichannel pipettor.

The plates can be tilted at a 30-deg angle so that the sharp ends of the pipet tips

can reach the medium at the bottom of the wells.

7) Replenish the wells with 200 L fresh medium. This precaution is taken to ensure

that the volume of medium is consistent throughout the wells. There may be

some loss in medium in the peripheral wells during the 3-day incubation.