TABLE 1

FCS and PCH analysis of HEK293 cells expressing YFP- and GFP-tagged biogenic amine receptors

Known monomeric (CD-86) and dimeric (CD-28) receptors are included as controls. M₁/GFP and β₂-AR/GFP were coexpressed with a 3-fold excess of untagged/nonfluorescent receptors (e.g., M₁/GFP + M₁/no tag 1:3). FCS τ_D2 values for GPCR diffusion within the plasma membrane are reported in milliseconds and represent the average dwell time of the receptor in the observation volume. Diffusion coefficients (μm²/s) were calculated using Eq. 3 (in the Materials and Methods). The 2D FCS molecular brightness values were determined by dividing the photon count rate by the number of molecules (as in Eqs. 4 and 5 in the Materials and Methods). The 3D PCH analysis was performed using the PCH module in the Zeiss Aim 4.2 software package by fitting the data to a one-component model (for a single population of fluorescent species) and the resulting reduced χ² values are reported. Data represent the mean ± S.E.M. for the number of cells as indicated.