Shalom.The BW Reporter System enables to study receptor-ligand interactions. It is extremely useful in cases where the ligand of a specific receptor is unknown or in cases where the experiments with the endogenous ligand are technically difficult. This method is sensitive and specific to an individual receptor, easy to operate and reproducible.
Demonstrating the procedure will be Shira Kahlon, a postdoc for my laboratory. The day before transfection, plate 10 plates with 10 milliliters of 100, 000 BW5147 cells, and supplement an RPMI. Then place 100 micrograms of the pcDNA3 plasmid in a tube.
Add sodium acetate at pH 5.3 to the tube. After this, add 2.5 volumes of 100%ethanol to the plasmid mixture. Then incubate the plasmid mixture overnight at minus 20 degrees Celsius.
24 hours after plating the BW cells, collect the cells in two 50 milliliter tubes. Then centrifuge the cells at 515 g's for five minutes, and discard the supernatant. Resuspend the peloton RPMI without additions and repeat the centrifugation.
Resuspend the resulting pellet in 1 milliliter of RPMI without additions. Transfer the resuspended pellet to a 4 centimeter cuvet on ice. Centrifuge the plasmid that underwent ethanol precipitation.
Then wash the plasmid with one milliliter of 70%ethanol. And repeat centrifugation for another 20 minutes. Remove all of the ethanol, and allow the pellet to dry partially.
Then resuspend the pellet in 100 microliters of preheated RPMI, without additions. After this, add the resuspended plasmid to the cells in cuvet. Incubate the cells on ice for five minutes.
Next, electroporate the cells. Then transfer the electroporated cells to a 50 milliliter tube. Add 50 milliliters of complete medium to the tube and centrifuge for five minutes, at 515 g's.
Discard the supernatant, and resuspend the cells in 50 milliliter of complete medium. Then plate the cells in 24-well culture dishes, and incubate the cells for 48 hours. After this, add one milliliter of complete medium, supplemented with 10 milligrams per milliliter of G418 to each well.
48 hours later, discard one milliliter of the upper volume of each well. Then add one milliliter of complete medium supplemented with five milligrams per milliliter of G418 to each well. Use 50, 000 transfected BW cells with their desired targets, in a single well of a 96-well plate.
Incubate the plate for 48 hours. Then freeze the plate at minus 20 degrees Celsius. Coat an ELISA plate with anti-mouse IL-2 antibody.
Place 05 micrograms of anti-mouse IL-2, in a volume of 50 microliters of PBS in each well. Incubate the coated plate overnight at four degrees Celsius. Use a brief incubation period at 37 degrees Celsius, to thaw the frozen BW cells, that were previously incubated with their targets.
Then centrifuge the plate, and transfer 100 microliters of the supernatant from each well to the pre-coated ELISA plate. And incubate at 37 degrees Celsius for two hours. After this add biotin anti-mouse IL-2 to the ELISA plate.
Then after incubation and washing, add HRP conjugated streptavidin to the plate. After incubation and washing, add 100 microliters of the TMB substrate solution, to each well of the ELISA plate. Finally read the ELISA plate at 650 nanometers.
In this example, CLL cells were pre-incubated with different anti-CD20 antibodies. And then co-incubated with transfected BW cells which express the Fc receptors CD16, before being analyze with ELISA. After analysis with ELISA the results were quantified.
The optical density levels, were converted into mouse IL-2 concentrations, based upon the standard curve. In an additional experiment, different doses of anti-CD20 antibodies were pre-incubated with CLL cells. And then incubated with the transfected BW cells.
The particle contains four main parts. Cloning, transfection of BW cells, activation and ELISA. Each of the parts should be verified independently.
Following this procedure, additional experiments can be performed with cells which express the endogenous receptors, such as killing acids with NK cells to study interactions with NK cell receptors. We have used this system to discover new ligands of NK cell receptors. For example, hemagglutinin is a ligand of NKp46, and PVL is a ligand of TG.
