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A sample, called a "dot blot", is the result of harvesting the contents of a microtiter plate onto a glassfiber filter. The experiment is a cell proliferation assay. The purpose of which was to ascertain the effect of different chemicals on live cells. Radioactive Thymidine is added to all the wells on the microtiter plate at the end of the experiment, and incubated for 6 hours. The radioactive Thymidine will be incorporated by all living cells as they divide. Hence, the radioactivity in a well correlates to the number of live cells in that well. The microtiter plate contains 96 wells in an 8 x 12 format. After harvesting the 96 samples onto the filter, the filter is dried. It is then necessary to examine each of the 96 spots to determine what, if any, radioactivity is present.

Traditionally, this would have been done in several ways.

1. Each spot would be cut out and placed in a scintillation vial, a liquid scintillation cocktail added, and the vial counted in a Liquid Scintillation Counter. This method was tedious, slow, time consuming, and expensive due to the cost of the vials and the cocktail, but it was accurate.

2. The dried filter could be placed in a Direct Beta Counter, the Matrix96 or 9600 from Packard Instrument Ltd, and all 96 wells counted simultaneously. The advantage is the ease of sample preparation, fast counting, and the data reduction is easy with the in-built computer. The disadvantage is the large initial cost of the equipment, and the relative low counting efficiency leading to longer counting times for low activity samples

3. The dried filter would be placed in a plastic bag, or a microplate type holder, and have scintillation cocktail added. The sample would then and counted in an appropriate instrument such as the Packard TopCount, Wallac MicroBeta or BetaPlate. The advantage of this method was higher counting efficiency. The disadvantages is through-put was slower, sample preparation was time consuming and costly, and high equipment cost.

4. Autoradiography or Fluorography as described in Experiment 1.

The Dot Blot attached (³H Thymidine DNA) was prepared by melting the EA-Wax into it. The sample was allowed to cool, and then placed with the preflashed film between two sheets of aluminium foil, in a cassette, and placed in a -70⁰C freezer for 16 hours.

The radioactivity ranged from a maximum of 4000cpm down to less the 100cpm. The data from the top row of spots was acquired by cutting out the row and then to cut each individual spot into a scintillation vial, and the vials counted in a scintillation counter. The counts per minute for each spot are also shown.

Spots 10, 9, and 8 had 163, 328 and 287 cpm respectively. The exposure time was 16 hours. If processed in the normal way, in this laboratory using autoradiography, the time taken would have been 3 to 4 weeks.

1. No expensive equipment is required.

2. The cost per sample is reduced.

3. The end result is obtained accurately and quickly.

4. Sample preparation is simple.

The main reason for this experiment was to demonstrate the sensitivity of Enhanced Autoradiography. The capacity to detect 300dpm of Tritium(³H) on film in a period of 16 hours is unique.

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