In phase-separated GPMVs from RBL-2H3 cells at 10°C, 488neg- and 594neg-SMs as well as 594neg-DSPC preferentially partitioned into Lo-like domains, whereas 594neg-DOPC partitioned into Ld-like domains. (A) Representative still images taken from simultaneous two-color video sequences. First and second rows, 594neg- and 488neg-SM exhibited partitioning complementary with Ld-domain marker molecules. Third and fourth rows, 488neg- and 594neg-SMs and 594neg-DSPC exhibited similar partitioning. (B) Ratio of the mean fluorescence signal intensity in the Lo-like domain versus that in the Ld-like domain (I_o/I_d; mean ± SE; n = 17, 22, 17, and 18 GPMVs from top to bottom). The same probe exhibited slightly different ratios because of variations in the leak signal from the other dye molecule in the pair. The mean ratios for each probe under various labeling conditions used here are as follows (mean ± SE, n = number of GPMVs examined): 488neg-SM, 4.5 ± 0.2 (n = 57); 594neg-SM, 4.5 ± 0.3 (n = 34); 594neg-DSPC, 4.5 ± 0.4 (n = 18); 594neg-DOPC 0.2 ± 0.02 (n = 22); and Bodipy FL-PC, 0.2 ± 0.02 (n = 17).