TABLE 1.

Diffusion (FCS) and molecular brightness (PCH) of fluorescent tags in solution or in the cytoplasm, and fluorescence-tagged receptors on the plasma membrane

HEK293 cells expressing a known monomeric receptor (CD-86) were used as a control to determine the molecular brightness of a monomer (CD-86/GFP). The molecular brightness of a dimer was determined using a tandem GFP tag (CD-86/GFP-GFP). Diffusion reported in milliseconds represents the average dwell time of the receptor in the observation volume. Diffusion coefficients (micrometers squared per second) were calculated using a 2D model for the lateral diffusion of receptors within the plasma membrane. PCH molecular brightness values are reported as CPSM. Reduced χ² values are reported for the PCH data fit to a one-component model for a single, homogeneous population of monomers or homodimers. Data represent the mean ± S.E.M. for the number of cells examined (N).

Fluorescent Tags and Labeled Receptors	FCS Diffusion		PCH Brightness	Reduced	N
	ms	μm2/s	CPSM	χ2
2C-Fab-GFP (S)	—a	—	8795 ± 145	1.14 ± 0.03	35
mGFP in HEK293 cells (C)	—	—	9075 ± 287	1.07 ± 0.02	25
CD-86/GFP in HEK293 cells (PM)	37.0 ± 2.0	0.61 ± 0.03	10,333 ± 364	1.09 ± 0.07	12
CD-86/GFP-GFP in HEK293 cells (PM)	37.4 ± 2.2	0.60 ± 0.04	19,691 ± 530	1.33 ± 0.10	10
5-HT2C/GFP in HEK293 cells (PM)	40.4 ± 1.9	0.56 ± 0.03	18,330 ± 674	1.19 ± 0.07	15
2C-Fab-GFP–labeled 5-HT2C receptors in HEK293 cells (PM)	48.7 ± 2.2	0.46 ± 0.02	18,783 ± 384	1.51 ± 0.12	20
2C-Fab-GFP–labeled 5-HT2C receptors in choroid plexus epithelial cells (PM)	42.6 ± 1.8	0.53 ± 0.02	18,466 ± 361	1.55 ± 0.22	10

Cytoplasm, C; plasma membrane, PM; solution, S.

^aNot calculated.