Fig. 4: 4Ig-B7-H3 dimerization detected in fixed cells using FRET-FLIM.
a The scheme for fluorescence lifetime changes observed in a monomeric versus dimeric example, where monomeric GFP lifetime is measured at 2.6 ns compared to GFP lifetime measurements under homo-FRET conditions that results in a reduced lifetime as energy transfer occurs between the two fluorescent proteins when their localization is less than 10 nm apart. b Representative images showing fluorescence lifetime of monomeric GFP exogenously expressed in an empty vector, or C-terminally linked to CD276 (4Ig-B7-H3). c A representative curve-fit for the lifetime decay of CD276-mEGFP using a bi-exponential fit. Calculated IRF (red), Overall decay (green), Fitted Curve (black), Residual pixels (green) are plotted below. d The average lifetime of GFP fluorescence determined for individual HeLa CD276-/- KO cells transiently transfected with Vector-mEGFP or CD276-mEGFP, where a mono-exponential fit was the best fit for Vector-mEGFP (average lifetime 2.62 ± 0.1141 ns) and a bi-exponential fit was the best fit for CD276-mEGFP (2.57 ± 0.1658 ns and 1.33 ± 0.0939 ns). Bars represent the mean (± standard deviation) lifetimes measured for 10 individual cells over three biological replicates.
