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Use of BRET2 luminescence cell-based assays with LEADseeker Multimodality Imaging System

Key words: luminescence • BRET2 • LEADseeker • DeepBlueC• cell-based assays


Competitive pressure is forcing organizations to continually increase screening throughput. Yet accuracy and sensitivity remain vital concerns, particularly for common screens such as luminescence cell-based assays. LEADseeker™ Multimodality Imaging System was developed to meet these demands by combining excellent detection with the ability to image whole plates in less than one minute.

This application note describes the use of luminescence emissions filters, which were defined and characterized using the BRET2™ Demonstration Assay Kit. The data demonstrate that BRET2 cell-based assays perform well in conjunction with the chemiluminescent readout of LEADseeker Multimodality Imaging System. Together, the filters, demonstration kit, and assay system support organizations looking to add high and ultra-high throughput luminescence cell based assays to their screening programs.


Materials
Products used

LEADseeker Multimodality Imaging System 18-1140-71

Luminescence filter configuration for Bret2 consists of: 11-0013-49

Emission filter (Luciferin donor) 470 nm, bp 50 nm

Emission filter (YFP acceptor) 535 nm, bp 45 nm


Other materials required
BRET2 Demonstration Assay Kit (PerkinElmer)

DeepBlueC™ (PerkinElmer)

384-well plates (non-treated, non-sterile, polystyrene, white)


Method
1. Reagent preparation
1.1 Prepare reagents as recommended by the kit manufacturer. Reagents, cell extracts (three vials) and buffer (four vials) should be thawed on ice.

1.2 Preparation of DeepBlueC

1.2.1 Add 25 µl ethanol to the DeepBlueC vial (final concentration = 1 mM).

1.2.2 Prepare a 100-fold dilution by adding 1.98 ml of buffer to 20 µl of the solution prepared in 1.2.1 (final concentration = 10 µM).

1.2.3 Place the solution on ice, protected from light.

2. Assay procedure
(Note: The assay was set up in triplicate and both 10 µl and 2 µl cell-extract assays were performed in the same microplate.)
2.1 Add 15 µl BRET2 buffer to wells A1–A3, B1–B3, and C1–C3.

2.2 Add 23 µl BRET2 buffer to wells A4–A6, B4–B6, and C4–C6.

2.3 Add 25 µl BRET2 buffer to wells D1–D3.

2.4 Dispense 10 µl of each cell extract as follows:

2.4.1 A1–A3: clear cap (non-transfected cells)

2.4.2 B1–B3: blue cap [9Rluc(h) + GFP2 transfected cells -negative BRET control]

2.4.3 C1–C3: green cap [GFP2 - Rluc(h) fusion transfected cells - positive BRET control]

2.5 Dispense 2 µl of each cell extract as follows:

2.5.1 A4–A6: clear cap (non-transfected cells)

2.5.2 B4–B6: blue cap [9Rluc(h) + GFP2 transfected cells -negative BRET control]

2.5.3 C4–C6: green cap [GFP2 - Rluc(h) fusion transfected cells - positive BRET control]

2.6 Dispense 25 µl 10 µM DeepBlueC (see Reagent Preparation Protocol, above) to all wells.

2.7 Image on LEADseeker Multimodality Imaging System for 60 s (within 60 min following dispensing).


Results
The BRET2 demonstration assay uses the luminescence emission signal from the donor and the luminescence energy transfer signal from the acceptor to generate the two sets of data used for the final analysis. The filters developed for the system are configured for donor emission at 470 nm and 535 nm for acceptor emission.

Table 1 shows the raw signal data generated on the LEADseeker Multimodality Imaging System for the donor and acceptor channels. Signal to noise was calculated using the following equation:

S:N values for all conditions in both assay volumes was < 10:1 (comparable with pack leaflet data), indicating that the signals generated were sufficient for final data analysis.

In the final data analysis, we ratioed the data obtained from the two channels of the raw signal analysis experiment and compared the ratios for the positive and negative control cell extracts (Fig 1).



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