PROCEDURE (nonsterile techniques) - at the end of the 16-hour exposure

ERDC TN-DOER-C10

March 2000

PROCEDURE (nonsterile techniques) - at the end of the 16-hour exposure.

1) Turn on the luminometer to warm up and run its self-test program. The injectors

of the luminometer will be primed during the 15-minute interval when the cells

are being lysed. Program the luminometer to inject 100 L each of Substrate A

and Substrate B simultaneously and to integrate luminescence readings over

2 seconds for each well.

2) Place an aliquot each of Substrate A, Substrate B, and Cell Lysis Buffer (1x) in

the water bath (room temperature).

3) Take out the two plates from the incubator to the hood.

4) Pipet off the cell medium in both plates using a multichannel pipettor, being very

careful not to remove any cells. 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.

5) Pipet 200 L of PBS (1x) into each well to wash the cells. Pipet off the PBS. The

cell medium and PBS rinse are treated as TCDD waste material and are

transferred to an appropriate container for later treatment and disposal.

6) IMPORTANT: Check each well to make sure that the PBS medium is

completely removed.

7) Add 40 L of cell lysis buffer to each well.

8) Place each 96-well plate onto the microplate adaptor holder of a vortex.

9) Set to vortex at number 4. Turn on switch and vortex for 15 minutes. In the

meantime, the luminometer can be primed (see Section IV.C, "SETTING UP

THE LUMINOMETER").

10) Check the wells under the microscope to see if the cells have been lysed. The cell

debris will clump up and float in the cell lysate.

11) Centrifuge plates at 5,000 rpm for 5 minutes. This step is taken to spin down the

cell debris (which can interfere with the luminometer reading) to the bottom of

the wells.

12) With a multichannel pipettor, transfer 25 L of the lysate in the first column of

cell lysate into the corresponding first column of wells of a 96-well luminometer

plate (opaque, nonsterile).

13) Rinse the pipet tips with PBS twice before proceeding with the next column of

wells.

14) Transfer the second 25 L of lysate from the second column of cell lysate to the

first column of cells in the 96-well luminometer plate.

15) IMPORTANT: Remember always to combine the 25-L lysates from two

consecutive wells into the 96-well luminometer plate. The lysates from the two

96-well plates should finally be transferred into one luminometer plate. See

following diagram for example.