Automated Measurement of Luciferase Luminescence Using Perkin Elmer Top Count

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The TopCount is a scintillation counter using a set of photon-multiplier tubes to measure luminescence of samples in 96-well microtiter plates (Fig. 3). It automatically cycles through a stack of up to 20 plates, which are held on top of the instrument, and returns luminescence data in text or Excel files. Using the TopCount, luciferase activity of 960 plants free-running in constant darkness can be measured every 45 min for several days. LED arrays can be fixed alongside the stacks and reflector plates (see Note 2) inserted between microtiter plates to reflect light from the side, down into the wells. Using this setup, luciferase activity can also be measured under constant light or in light-dark cycles. The usefulness of the TopCount is limited by the amount of light that can be reflected into microtiter plates. Plants displaying interesting circa-

Fig. 2. Normalized luminescence of the circadian regulated promoters CCR2, CCA1, and CAB fused to the luciferase reporter gene. Luminescence levels were quantified using MetaMorph software from images captured using an ORCA II cooled charge-coupled device camera. Plants were entrained in 12 h light/12 h dark cycles and free-run in constant blue light. Data acquisition began at the start of the transfer to constant conditions.

Fig. 2. Normalized luminescence of the circadian regulated promoters CCR2, CCA1, and CAB fused to the luciferase reporter gene. Luminescence levels were quantified using MetaMorph software from images captured using an ORCA II cooled charge-coupled device camera. Plants were entrained in 12 h light/12 h dark cycles and free-run in constant blue light. Data acquisition began at the start of the transfer to constant conditions.

dian phenotypes can be rescued from microtiter plates, planted in soil, and allowed to set seed.

1. Sterilize and entrain seedlings as described in Subheading 3.3.

2. Melt 200 mL of MS solid media. In a sterile flow hood add 300 ^L to each well of a black 96-well microtiter plate (see Note 10). Allow to dry and wrap plates in plastic film. Plates can be used immediately or stored at 4°C.

3. Transfer seedlings with sterile forceps to each well. Hold the seedling between root and hypocotyl and insert the root into the agar plug (see Note 11).

4. Pipet 15 ^L of 5 mM luciferin into each well (see Note 8).

5. Take two sheets of TopSeal; cut 5 mm off all the way around one sheet. Stick the two sheets together, sticky side to sticky side, creating a 5-mm sticky border. This can be stuck to the plate without plants becoming stuck to the TopSeal. Using a hypodermic needle, pierce the TopSeal above each well to allow gas exchange.

6. Stick an individual barcode label to the right-hand side of the plate and wipe the top of the plate with WD-40 to prevent plates jamming onto each other in the stack.

7. Return the plates to the entrainment cycle and, at dusk on the seventh day, load into the TopCount.

Fig. 3. Pictorial representation of the method used to analyze circadian rhythms in Arabidopsis. Top left: Seedlings entraining to light cycle on MS plates. Bottom left: Seedlings in a black microtiter plate ready to be assayed. Center: Packard TopCount with light-emitting diode arrays attached to the sides of the stackers. Top right: Excel worksheet program, TopTemp. Bottom right: Assayed plants transferred from microtiter plates to soil and allowed to flower.

Fig. 3. Pictorial representation of the method used to analyze circadian rhythms in Arabidopsis. Top left: Seedlings entraining to light cycle on MS plates. Bottom left: Seedlings in a black microtiter plate ready to be assayed. Center: Packard TopCount with light-emitting diode arrays attached to the sides of the stackers. Top right: Excel worksheet program, TopTemp. Bottom right: Assayed plants transferred from microtiter plates to soil and allowed to flower.

8. Set TopCount to run indefinitely measuring luminescence; check periodically that the machine is still working and that plates have not become jammed. For constant light experiments, the lights are set to come on at dawn and remain on, using a commercial timer.

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