The overall goal of this aggregation assay is to assess the binding of Notch receptor with trans-ligands and its inhibition by cis-ligands. This method can help answer a key question, How will genetic or pharmacological factor affect the binding of Notch receptor to its ligand, and to alter the signaling? The main advantage of this technique is that it allows to evaluate the trans-and cis-binding of Notch receptor with its ligand.
Interestingly, without involving the overlapping effect of endogenous ligand. Begin the assessment by spinning down signal-receiving cells and resuspending them in Schneider's Medium. Count the cells manually using a hemacytometer.
Plate five times 10 to the fifth S2 and stable S2-Notch cells in each well of a six-well plate, in one milliliter per well of Schneider's Medium. Next, add 7.5 micrograms of double-stranded RNA to each well, and incubate the cells at 25 degrees Celsius for 24 hours. Add 0.7 millimolar of copper sulfate to induce the expression of Notch, and incubate the cells at 25 degrees Celsius for three days.
After incubation, count the signal-sending cells manually using a hemacytometer. And plate five times 10 to the sixth stable cells in each well of a six-well plate, in one milliliter per well of supplemented Schneider's Medium. Then add 0.7 millimolar of copper sulfate to induce the expression of ligands, and incubate the cells at 25 degrees Celsius for three hours.
Harvest the double-stranded RNA-treated S2-control and S2-Notch cells by gentle pipetting. And plate 2.5 times 10 to the fifth cells per well in a 24-well plate. Add five times 10 to the fifth stable S2-Delta or S2-Serrate Tomato cells to a total volume of 200 microliters of supplemented Schneider's Medium.
Place the plate on an orbital shaker at 150 rpm. After one minute, mix the contents of each well, and take 20 microliters out to count the number of aggregates. Simultaneously, take a representative image under an inverted compound microscope using 10x magnification.
To visualize the cells, set the camera on the microscope, and connect with the software to acquire the image. Manually count the aggregates using a hemacytometer. Place the plate on the shaker.
Repeat the image acquisition and counting after five minutes and 15 minutes of aggregation. Perform trans-binding quantification by calculating the number of aggregates per milliliter between S2 cells and S2-Delta or S2-Serrate Tomato cells as a background control, and the number of aggregates per milliliter between S2-Notch and S2-Delta or S2-Serrate Tomato cells. Prepare the signal-receiving cells by plating five times 10 to the fifth S2 cells in each well of a six-well plate, in one milliliter per well of supplemented Schneider's Medium.
Add 7.5 micrograms of double-stranded RNA to each well, and incubate the plate at 25 degrees Celsius for 24 hours. After incubation, cotransfect the double-stranded RNA-treated cells for a total DNA concentration of two micrograms per well using a commercial transfection reagent. Incubate the transiently-transfected S2 cells at 25 degrees Celsius for 24 hours.
The next day, add 0.7 millimolar copper sulfate to induce the expression of Notch and the ligands, and incubate at 25 degrees Celsius for three days. Prepare signal-sending cells as described in the previous section. And finally, perform aggregation between signal-sending and signal-receiving cells, as described previously.
A sharp increase was observed in the number of aggregates between one and five minutes. Then the number of aggregates continued to increase until 30 minutes, at a slower rate. Images showing the aggregation at three timepoints demonstrate the EGFP double-stranded RNA-treated S2-Notch cells formed aggregates with S2-Delta cells, which increase in number with time.
In contrast, shams and double-stranded RNA-treated S2-Notch cells formed aggregates faster, and the aggregates were bigger and greater in number. Indicating that decreasing shams levels enhanced the trans-binding of Notch with Delta. Transfecting equal amounts of Notch and Delta expression constructs into S2 cells dramatically decreases the number of aggregates formed between these cells and S2-Delta cells.
Moreover, decreasing the amount of cis-Delta results in an increase in the number of aggregates. Indicating that in the range of Notch/cis-Delta ratios used in these assays, cis-Delta can inhibit the binding between Notch and trans-Delta in a dosage-dependent manner. Quantification of relative aggregation showed a comparable inhibition of aggregation upon EGFP and shams double-stranded RNA treatment.
Once all the reagents are prepared, this assay is straightforward and can be executed in a few hours.
