Excitation scans were generated using a Deltascan dual-wavelength fluorimeter from Photon Technology International (PTI; Birmingham, NJ) and the associated Felix software. Standard HEK293 cells were plated on 10 cm dishes and transiently transfected with 5 µg each of the soluble forms of GFP2, pHluorin and pHluorin2 using Fugene HD (Roche, Indianapolis, IN). Forty-eight hours post transfection, cells were washed with Hank’s Balanced Salt solution lacking calcium and magnesium ions and containing 2 mM EDTA. Cells were lifted from the plates using the same solution after a 10 minute incubation at 37°C and placed in a quartz cuvette with a stir bar. Excitation scans were performed between wavelengths 370 nm to 490 nm with the emission set at 510 nm. For the pH clamp experiments, cells were resuspended in a buffer containing 140 mM potassium chloride, 10 mM sodium phosphate with molar ratios of mono- and dibasic forms appropriate for the given pH and 30 mM nigericin (EMD Biosciences, Gibbstown, NJ). For the florescence plate reader studies, black, 96-well plates (Perkin-Elmer, Boston, MA) were loaded with 25,000 cells per well from the transfections described above and analyzed with dual excitation at 405 nm/8 nm and 485 nm/25 nm with a 535 nm/25 nm emission filter in an EnVision plate reader (Perkin-Elmer, Boston, MA). The narrow bandwidth of the 405 nm filter of 8 nm reduces the relative florescence compared to the larger bandwidth of the 485 nm filter. Despite this effect, the 405/485 nm ratios are still responsive to changes in intracellular pH.
For the hPTH1R endocytosis studies, HEK293 cells were sparsely plated into culture slides and transiently transfected with 100 ng of the hPTH1R-pHluorin2 construct using Fugene HD. Forty-eight hours post-transfection, cells were treated with either vehicle (acetic acid) or 100 nM parathyroid hormone containing amino acids 1 to 34 for 20 minutes. Cells were fixed with 3.7% paraformaldehyde and analyzed by confocal microscopy using a Radiance 2100 confocal microscope and the associated LaserSharp 2000 software (Bio-Rad Laboratories, Inc., Hercules, CA).
For the hPTH1R endocytosis studies, HEK293 cells were sparsely plated into culture slides and transiently transfected with 100 ng of the hPTH1R-pHluorin2 construct using Fugene HD. Forty-eight hours post-transfection, cells were treated with either vehicle (acetic acid) or 100 nM parathyroid hormone containing amino acids 1 to 34 for 20 minutes. Cells were fixed with 3.7% paraformaldehyde and analyzed by confocal microscopy using a Radiance 2100 confocal microscope and the associated LaserSharp 2000 software (Bio-Rad Laboratories, Inc., Hercules, CA).
