Day 2 (DOSING THE CELLS)

ERDC TN-DOER-C30

February 2003

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. This

volume of cell suspension will be sufficient for seeding two 96-well plates.

Example

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.

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.

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

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

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

suspension thoroughly before pipetting each plate.

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

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

III.

Day 2 (DOSING THE CELLS)

The cells are dosed with Ponasterone A standards (prepared in DMSO) and environmental

samples (prepared in iso-octane) on this day. The following setup is for testing 14 environmental

samples with 3 replicates each.

MATERIALS AND EQUIPMENT

Ponasterone A (10 mM) stock in DMSO, 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

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 min

for equilibration.

3) Turn on 37 C water bath.