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Page Title:
Protocol II. Performance of the Assay |
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 ERDC TN-DOER-C10
March 2000
Protocol II. Performance of the Assay
I.
PRELIMINARY.
Four days are required for performance of the assay. On Day 0, preferably a Monday or Friday,
cells are plated in two 96-well microtiter plates and incubated for 3 days. On Day 3, preferably the
later part of the afternoon, the cells are dosed with serial dilutions of the
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) standard and with the environmental extracts. The
cells are again incubated for another 16 hours to allow for enzyme (luciferase) induction. On Day 4, the
cells are lysed and the luciferase production is quantified using a luminometer.
Two 96-well microtiter plates are used for testing up to 24 environmental samples with 3
replicates each. One-half of a 96-well plate is allocated for the TCDD standards while the rest of
the wells are used for the environmental samples. The second 96-well plate will not be necessary
if testing fewer than five samples.
The time required to complete each daily task using the 96-well plates is described in the
following tabulation:
Time,
Task
hr
Day 0
1.0
Day 3
0.5
Day 4
1.0
Solution preparation/equipment setup
1.0
Total
3.5
II.
DAY 0 (PLATING CELLS).
A.
MATERIALS AND EQUIPMENT.
MEM.
Versene (1x, 0.2 g EDTA.4Na).
Geneticin (G418) (40 mg/mL) -1 aliquot.
Trypsin (1x) - 1 aliquot.
Trypan Blue (0.05 percent) - one aliquot.
Multichannel motorized pipettor (8 channels, 250 L).
96-well microtiter plate, flat bottom, sterile (Corning #25860).
Sterile pipet tips (250 L).
Pasteur pipets (9 in., autoclaved).
Hemacytometer and cover slip.
Inverted microscope.
50-mL sterile centrifuge tube.
50-mL sterile solution basins.
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