Grow cells on 150 mm dish to ~70% confluency

Wash twice with 10 mL PBS

Detach cells with 1.5 mL trypsin-EDTA + 10.5 mL media. Trypsinize for 5 minutes and check under microscope for complete detachment; tap dish if necessary

Count cells with hematocytometer and dilute collected cells to 164,000 cells/mL

Pipet 1 mL into each well of poly-D-lysine coated 12-well plate

Add 250 mL of 5x doxycycline for all desired concentrations to cells 14 hours before assay

Prepare > 300 uL ligand-containing media per well (e.g. 10 uM AngII) for stimulation

Remove media from plate wells by vacuum in cell culture hood

Add 300 uL ligand-containing media per well and return to incubator for 1 hour

Prepare saturating concentration of membrane-impermeable radioligand in media on ice. A ligand with high specific activity is preferred. For example, 30 uL 3H-AngII in 12 mL media. Prepare at least 4 mL per plate. Vortex briefly to mix

Take 1/3 of media with radioligand and add high concentration (orders of magnitude above radioligand concentration) of nonlabelled antagonist (e.g. 200 uM candesartan final concentration) for nonspecific binding measurement. Vortex briefly to mix

After 1 hour ligand incubation, place plates on ice and remove media by vacuum. Wash twice with 1 mL ice-cold PBS with MgCl2 and CaCl2 per well.

Strip ligand for 5 minutes with 2 mL acid wash buffer (0.2 M acetic acid, 0.5 M NaCl, pH 2.5) per well

Remove acid wash buffer by vacuum and wash 2-3 times with 1 mL ice-cold PBS with MgCl2 and CaCl2 per well.

Remove wash by vacuum. 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

Pre-chill 50 mL per plate PBS with MgCl2 and CaCl2 on ice and pre-warm 0.1% SDS + 0.5 N NaOH in 42 °C water bath

Remove radioligand media into dedicated radioactive liquid waste vial using P1000 on ice. Remove nonspecific binding media last

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. Check plates under microscope after last wash

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, shake for ≥ 15 minutes

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

Wipe vials with moist KimWipe and place in racks for scintillation counter. Place racks in counter and count using 3h_cpm_3_min protocol

Perform second count using different protocol flag on next day

Copy binding counts into Excel spreadsheet

Calculate nonspecific counts for each receptor concentration

Calculate specific counts for each condition by averaging duplicates and subtracting nonspecific count

Calculate percent internalization as percent reduction in specific counts for ligand-stimulated condition compared to control