TITLE: Whole-cell radioligand saturation binding
AUTHORS: Angus Li, Samuel Liu, Rennica Huang, Seungkirl Ahn, Robert J Lefkowitz

[DESCRIPTION]
This protocol details an experimental procedure used to generate results described in the manuscript Li, A., Liu, S., Huang, R., Ahn, S., & Lefkowitz, R. J. (2023). Loss of biased signaling at a G protein-coupled receptor in overexpressed systems.

[STEPS]
SECTION: Day 1
1. Grow cells on 150 mm dish to ~70% confluency
SECTION: Day 1
2. Wash twice with 10 mL PBS
SECTION: Day 1
3. Detach cells with 1 mL trypsin-EDTA + 11 mL media. Trypsinize for 5 minutes and check under microscope for complete detachment; tap dish if necessary
SECTION: Day 1
4. Count cells with hematocytometer and dilute collected cells to 164,000 cells/mL
SECTION: Day 1
5. Pipet 1 mL into each well of poly-D-lysine coated 12-well plate. Make duplicate plates for all conditions: one plate for binding and one for BCA assay
SECTION: Day 2
6. Add 250 mL of 5x doxycycline for all desired concentrations to cells 14 hours before assay
SECTION: Day 3 (BCA)
7. Aspirate media from BCA assay plates, wash each well with 1 mL cold PBS with MgCl2 and CaCl2, add 200 uL glycerol lysis buffer using repeater dispensing pipette. Shake in cold room
SECTION: Day 3 (BCA)
8. Fast cool centrifuge to 4 °C
SECTION: Day 3 (BCA)
9. Scrape each well with cell scraper, wiping with KimWipe and washing in cold PBS between wells. Pipet 150 uL lysate up and down 5x to wash wells and collect in Eppendorf tubes on ice
SECTION: Day 3 (BCA)
10. Centrifuge lysate for 10 minutes at 4 °C and 13,000 rpm
SECTION: Day 3 (BCA)
11. Collect 100 uL supernatant into new Eppendorf tubes on ice
SECTION: Day 3 (BCA)
12. Measure protein concentration using standard BCA assay protocol
SECTION: Day 3 (Binding)
13. Prepare saturating concentration of radioligand in media on ice. For example, 12.5 uL 3H-AngII in 5 mL media, or 4 uL 3H-olmesartan in 5.5 mL media. Prepare at least 5 mL per plate. Vortex briefly to mix
SECTION: Day 3 (Binding)
14. Take 1.5 mL media with radioligand per plate and add high concentration (orders of magnitude above radioligand concentration) of nonlabelled antagonist (e.g. 3 uL of 100 mM candesartan) for nonspecific binding measurement. Vortex briefly to mix
SECTION: Day 3 (Binding)
15. Dump media from plates into sink and invert plate onto paper towel to remove remaining liquid. Add 300 uL media with radioligand per well on ice. Use top two rows of plate for total binding duplicates and bottom row for nonspecific binding. Wait ≥ 1.5 hours on ice
SECTION: Day 3 (Binding)
16. Pre-chill 50 mL per plate PBS with MgCl2 and CaCl2 on ice and pre-warm 0.1% SDS + 0.5 N NaOH in 37 °C water bath
SECTION: Day 3 (Binding)
17. Remove radioligand media into dedicated radioactive liquid waste vial using P1000 on ice. Remove nonspecific binding media last
SECTION: Day 3 (Binding)
18. Wash wells with 1 mL cold PBS with MgCl2 and CaCl2 on ice and collect using P1000 into radioactive liquid waste vial. Wash 4 times total
SECTION: Day 3 (Binding)
19. Remove plates from ice and add 500 uL warm 0.1% SDS + 0.5 N NaOH per well. If there is precipitate in the solution, allow it to warm for longer. After adding, wait for ≥ 15 minutes
SECTION: Day 3 (Binding)
20. Collect samples from wells using P1000 and add to labeled scintillation vials. Add 300 uL of each radioligand media mix to two scintillation vials each for total counts. Add 5 mL scintillation cocktail (Lefko-Fluor) to each vial. Cap vials
SECTION: Day 3 (Binding)
21. Wipe vials with moist KimWipe and place in racks for scintillation counter. Place racks in counter and count using 3h_cpm_3_min protocol
SECTION: Day 3 (Binding)
22. Perform second count using different protocol flag on next day
SECTION: Analysis
23. Use GraphPad radioactivity calculator to calculate specific activity of radioligand. The counter efficiency of the beta counter is approximately 65%; check the latest CPA results for an exact value
SECTION: Analysis
24. Copy binding counts into Excel spreadsheet and label
SECTION: Analysis
25. Transfer protein concentrations from BCA assay into spreadsheet
SECTION: Analysis
26. Use GraphPad radioactivity calculator and data in spreadsheet to calculate receptor concentration in fmol/mg
SECTION: Analysis
27. Use GraphPad radioactivity calculator and total counts from radioligand media to calculate radioligand concentration in nM