The High-Throughput screening calcium fluorescence dual addition assay allows identification of novel small molecule ligands of a G protein-coupled receptor that signals through the intracellular calcium cascade. The main advantage of the dual addition assay is the detection of agonist and antagonist HTS in a single assay with the same cells provided the fluorescence signal last two minutes. This method can be applied to any GPCR that signals through the calcium cascade, which includes most of the arthropod neuron peptide family, a GPCRs.
For assay reproducibility, it is important to optimize the cell density, injection speed, and concentration of the known agonist for the second edition. Demonstrating the procedure will be Bianca Henriques-Santos, Post-doctoral research associate in my laboratory. Begin the calcium fluorescence assay by removing the spent medium from T-75 flask containing 70 to 90%confluent BMLK three cells.
Wash the cells with 10 milliliters of Dulbecco's phosphate-buffered saline or DPBS. After removing the DPBS, detach the cells using two milliliters of 0.25%trypsin ethylenediaminetetraacetic acid or EDTA and incubate for three to five minutes at 37 degrees Celsius. Add eight milliliters of selective medium and transfer the cell suspension into a 15 milliliter conical tube.
Before centrifuging the cell suspension at 1000 x g for three minutes. Discard the supernatant and resuspend the cell pellet in 10 milliliters of F-12K medium containing 1%fetal bovine serum or FBS and 400 micrograms per milliliter G418 sulfate. To determine the cell density of the suspension for further dilution, mix 20 microliters of cell suspension into 20 microliters of 0.4%trypan blue, and then load 20 microliters mixture into a cell counting chamber to be read by a cell counter for cell density.
Dilute the cell suspension using the same F-12K medium and make up the final volume to 15 milliliters at a density of four times 10 to the fifth cells per milliliter. To seed the cells in the Poly-D lysine or PDL coded 384 well plate. Add 25 microliters of the diluted cell suspension into 384 wells of the plate using a liquid handling system.
Incubate the plate overnight at 37 degrees Celsius and 5%carbon dioxide in the humidified incubator. On the next day, quickly invert the 90%confluent 384 well plate. To remove the spent medium and gently blotted on sterile paper towels two to three times to remove all the liquid from the plate.
Carry out the next steps in the soft dim light inside the biosafety cabinet. Next, add 25 microliters of the loading dye into each well using a liquid handling system by dispensing 12.5 microliters into each well with an aspiration and dispensing speed of 3.8 microliters per second. Repeat this pipetting step to reach a final volume of 25 microliters in each well.
Once done, cover the plate with aluminum foil to protect it from ambient light and incubate at 37 degrees Celsius in the carbon dioxide humidified incubator for 30 minutes. After equilibrating the covered plate for another 30 minutes, the cells are ready for high throughput screening or HTS. Next, centrifuge the drug plate at 1200 x g in a plate centrifuge for one minute.
Transfer the 10x agonist peptide solution of Rhimi-K-1 into a 150 milliliter auto-friendly reservoir. In the plate reader, click manage protocols. Then in the endpoint fluorescent intensity protocol, select Flow Forte pre-read, click start measurement to measure the background signal for the entire HTS assay.
Insert the cell plate into the plate reader. In the instrument panel, click on a random well on the plate then click on new focus height. Click on focus adjustment.
Click on gain adjustment. Assign it as 5%to 10%of the maximum measurable fluorescence value. Click start adjustment.
Click start measurement to read the whole plate for the background fluorescence signal in relative fluorescence units or RFU. While the plate is reading, click current state to see the real time reading value. For the dual-addition assay, pipette 10 microliters of the drug solution up and down three times using the liquid handling system and aspirate 1.5 microliters from each well of the drug plate at an aspiration speed of 1.0 microliters per second.
Dispense 0.5 microliters of the compounds into the cell assay to reach a final concentration of two micromolar in 0.2%dimethyl sulfoxide or DMSO. After adding the screening compounds, place the assay plate immediately into the plate reader. Click the preset reading program and read the plate in the forward and reverse reading directions.
Dispose of one microliter of drug solution remaining in the tips by immersing the tips into a 150 milliliter waste reservoir containing approximately 50 milliliters of DPBS. Incubate the screening compounds with the cells for a total of five minutes, including the time inside the plate reader in the biosafety cabinet with the lights off. Next, add three microliters of the agonist peptide Rhimi-K-1 from the reservoir into the assay plate using the liquid handling system using 384 12.5 microliters tips.
Place the plate into the plate reader immediately. Click the preset reading program and read the plate in the forward and reverse reading directions. Once done manually calculate the Z'factor for the quality control of each plate assay.
Manually select the hit molecules from the heat maps on the online HTS data platform and calculate the normalized percent activation or NPA and inhibitory activity, or I0 for the agonist hits and antagonist hits. An in-house drug plate SAC2-34-6170 composed of 320 random small molecules was used for demonstrating this HTS assay. The HTS had excellent assay quality with a Z'factor of 0.7 reflecting that the assay quality independent of the tested compounds.
It is essential to read the plate immediately after adding the agonist because the fluorescence signal is short-lived. After this procedure, the identified hit molecule should be validated through dose response assays and tested in cells that do not express the target GPCR to exclude off-target hits.
