β-Arrestin Assay Protocol

GPCR activation sets in motion a number of second messenger cascades that culminate in a physiological response after ligand binding. Cells expressing a calcium-sensitive receptor are pre-loaded with a calcium-sensitive dye before being treated with the chemical. The receptor stimulates the release of intracellular calcium, resulting in an increase in dye fluorescence. The Calcium Flux Assay can be used to track the activation of Ga_q-coupled receptors and recombinant promiscuous G protein receptors in cells that overexpress GPCRs.

Fig.1. Schematic diagram showing G protein-coupling mechanisms. (Sharan & Catherine, 2017)

Cell Thawing

1. Warm the growing media (without antibiotics) for 15 minutes in a 37°C water bath.
2. Fill a T75 flask with 15 mL of growth media and place it inside a sterile tissue culture hood. Set aside for Step 6. At this time/stage, DO NOT add selective antibiotics to the growth media.
3. Thaw the cryovials in dry ice immediately after removing them from -80°C or liquid nitrogen vapor storage.
4. Thaw the cells in cryovials in a 37°C water bath for 30 seconds to 1 minute, or until only small ice crystals remain and the cell pellet is almost entirely thawed. Freshly thawed cells should not be centrifuged or vortexed.
5. Spray and wipe the vials' external surfaces with 70% ethanol to decontaminate them. In a tissue culture hood, place the vials.
6. Gently transfer the thawed cells to the pre-filled T75 flask using a sterile pipette. Incubate the flask at 37°C with 5% CO₂ in the air.
7. Keep the cells in the culture until they are 70% to 80% confluent. Then follow the directions for Cell Recovery. If the cells are below this confluency, do not split them; otherwise, growth difficulties may arise.

Cell Recovery

1. Warm the growing media for 15 minutes in a 37°C water bath. DO NOT use antibiotics for selection in the growth media.
2. Place the T75 flask in a sterile tissue culture hood after removing it from the tissue culture incubator.
3. Remove the media from the T75 flask with gentle aspiration.
4. Fill the T75 flask with 5 mL PBS and gently rock it back and forth to rinse the cells.
5. Remove PBS from the flask with gentle aspiration.
6. Fill the flask with 1 mL of 0.25% Trypsin-EDTA. To ensure that the flask's inner surface is uniformly covered with trypsin, gently move it back and forth.
7. Incubate the flask for 2 to 3 minutes at 37°C with 5% CO₂, or until the cells have detached.
8. Take the flask out of the incubator and look at the cells under a microscope to make sure they're detached. If required, touch the flask's edge gently to separate cells from the surface.
9. Slowly pipette 4 mL of growth media into the separated cells in the T75 flask using a pipette. Rinse the cells gently with the reagent using a pipette. The trypsin in the cell suspension will be neutralized as well.
10. Gently pipet the cell suspension up and down many times to create a single, clump-free cell suspension.
11. Using a hemocytometer or an automated cell counter, count the number of viable cells. Calculate the number of cells and the amount of cell suspension needed to seed a T75 or T225 flask.
12. Fill a new T75 or T225 flask with 5 mL growth media, then add the cell suspension (volume determined in Step 11). To attain a final volume of 15 mL for a T75 flask or 45 mL for a T225 flask, add an additional volume of growth media.
13. Place the flask in a tissue culture incubator and keep the cells at 37°C with 5% CO₂.

Cell Freezing

1. Place T75 (or T225) flasks in a sterile tissue culture hood after removing them from the incubator.
2. Aspirate the media from the flasks slowly.
3. Fill each T75 flask with 10 mL PBS (or 15 mL for a T225 flask) and gently rock the flask back and forth to rinse the cells.
4. Remove PBS from the flask with gentle aspiration.
5. Fill T75 flasks with 1 mL of 0.25% Trypsin-EDTA (or 3 mL to T225 flasks).
6. Gently rotate the flask back and forth to ensure that Trypsin-EDTA is evenly distributed throughout the flask's inner surface.
7. Incubate the flasks for 2 to 3 minutes at 37°C with 5% CO₂, or until the cells have detached.
8. Take the flask out of the incubator and examine it under a microscope to make sure the cells have separated. If required, touch the flask's edge gently to separate cells from the surface.
9. To neutralize the trypsin, add 5 mL of cell culture media to each T75 flask (or 15 mL to each T225 flask).
10. Gently pipet the cell suspension up and down many times to create a single, clump-free cell suspension.
11. Place the cell suspension in a 15 mL conical centrifuge tube from the T75 flask. Transfer cells to a 50 mL conical centrifuge tube if using a T225 flask. If necessary, top up the flasks with additional cell culture media (e.g. 3 mL for T75 flasks or 5 mL for T225 flasks). Rinse the flask and transfer it to the conical tube to collect any residual cells. To break up clumps of cells, slowly pipette up and down numerous times.
12. Calculate the cell concentration in the suspension.
13. Centrifuge the collected cells for 4 minutes at 200g.
14. Discard the supernatant after centrifugation, being cautious not to disrupt the cell pellet.
15. Resuspend the cells to the required concentration (e.g. 2×10⁶ cells/mL) based on the total cell population estimated in Step 12.
16. Pour 1 mL of the cell suspension into each of the 2 mL cryogenic tubes that have been labeled. Tightly close the tubes.
17. Freeze cells overnight in a dedicated cell freezer at -80°C at a controlled rate of -1°C/minute. Vials can be kept in the -80°C freezer for up to two weeks for short-term storage.
18. Place the vials in the liquid nitrogen vapor phase for long-term storage.

β-Arrestin Assay

1. Cell preparation and plating
   1.1 Separate the cells and resuspend them in new medium.
   1.2 Take an aliquot of cells from the flask and calculate their density.
   1.3 Depending on the number of cells necessary for the desired number of samples to be run in the assay, transfer an appropriate volume of the cell suspension to a conical tube. To pellet cells, centrifuge at 300g for 4 minutes at room temperature.
   1.4 Carefully remove the medium with a 10 mL pipette without disturbing the cell pellet by decanting the supernatant.
   1.5 In the cell plating reagent, resuspend the cell pellet. Resuspend the pellet in an appropriate volume based on the number of cells in the pellet from Step 1.5 to reach the required cell concentration.
   1.6 Transfer the cell suspension to a clean reagent reservoir. Using a multichannel pipette, transfer 80 L of the cell suspension into each well of a 96-well test plate.
   1.7 Incubate the test plate at 37°C with 5% CO₂.
2. Compound Preparation
   In dilution buffer, prepare an 11-point series of 3-fold agonist serial dilutions in a separate dilution plate or vials. Each dilution's concentration should be 5× that of the final screening concentration.
   In dilution buffer, prepare an 11-point series of 3-fold antagonist serial dilutions in a separate dilution plate or vials. Each dilution's concentration should be 10× that of the final screening concentration.
   In dilution buffer, prepare an 11-point series of 3-fold anti-ligand antibody serial dilutions in a separate dilution plate or vials. Each dilution's concentration should be 10× that of the final screening concentration.
3. Detection
   Please refer to the β-arrestin detection kit datasheet to determine the correct detection kit to use before proceeding to the next step.

Reference

1. Sharan, S., Catherine, H. Potential of GPCR-Targeting Insecticides for Control of Arthropod Vectors: Volume 2: GPCRs and Ion Channels. 2017.