PROCEDURE (nonsterile techniques) - at the end of the 48th-hour exposure time point

ERDC TN-DOER-C30

February 2003

1) Connect the Dynex MRX plate reader to the computer and turn the reader and the

computer on.

2) Open the Revelation program, and open the assay file. The assay file should use

the following parameters:

Filter = 405 nm

Temperature = 37 C

Read at 1-min intervals for 15 min

3) Start a trial blank run to be sure cable connections and assay setup are correct.

PROCEDURE (nonsterile techniques) - at the end of the 48th-hour exposure time point.

1) Place an appropriate amount of ONPG substrate and Buffer A (with

-mercaptoethanol) in a 37 C water bath, along with an aliquot of cell lysis

buffer (1x).

2) Take out the plate from the incubator to the hood.

3) Pipet off the cell medium in plate 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.

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

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

removed.

5) Add 100 L of cell lysis buffer to each well.

6) Place the plate onto the microplate adaptor holder of a vortex.

7) Shake plates for 10 min at setting number 2. In the meantime, the

spectrophotometer can be set up.

8) 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.

9) Centrifuge plates at 5,000 rpm's for 5 min. This is done to spin down the cell

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

the wells.

10) With a multichannel pipettor, transfer 50 L of the lysate into the corresponding

wells of a clean 96-well plate.

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

wells.

Be sure that no bubbles are generated during the sample transfer; bubbles will

interfere with the spectrophotometric measurements.

12) When all lysates have been transferred, add 110 L of the prewarmed Buffer A

with -mercaptoethanol to each well.

13) Cover the plate and place in the incubator for 5 min at 37 C.