(A–B) Schematic illustration showing the process to deconvolve intermolecular and intramolecular FRET. In brief, tagged VRC01-IgM-BCRs were diluted by WT VRC01-IgM-BCRs to minimize intermolecular FRET. In the situation that two tags are in the same plasmid for expressing dually tagged BCR (A), most of the observed FRET signal will be intramolecular FRET. In the situation that two tags are in distinct two plasmids for expressing two types of singly tagged BCR (B), respectively, most of the observed FRET signal will be intermolecular FRET. (C–F) Dequenching FRET to measure the FRET efﬁciency between CoA 488 and ReAsH in 293T cells expressing tagged VRC01-IgM-BCR activated by V51 monomer antigen. Intramolecular FRET between CoA 488 and ReAsH in the same individual VRC01-IgM-BCR molecule (C) and intermolecular FRET between CoA 488 and ReAsH in two neighboring VRC01-IgM-BCR molecules (D) were shown. Intramolecular FRET (E) and intermolecular FRET (F) between CoA 488 and ReAsH without dilution were also shown. Cells with equal donor intensity and equal acceptor intensity were used for FRET analysis. Data are from at least 21 cells over three independent experiments. Two-tailed t-tests were used for the statistical comparisons. ***p<0.001; **p<0.01.