Fig. 2: The verification of stability, sensitivity, reversibility, and linearity of biosensors in SH-SY5Y cells.
From: Visualization of RIM-BP2’s crane-like function in neuronal vesicle transport using FRET
A The stimulation protocol for biosensors validation. Axonal regions were aligned between bipolar electrodes. The red arrow indicates the onset of stimulation, and the red line represents the duration of the stimulation. B, C Verification of biosensors stability. B FRET channel fluorescence images of SH-SY5Y cells transfected with BKTS and RKTS, and YPet/ECFP FRET ratio images of the red dashed area processed by K-means cluster analysis at 0 s and 300 s in the resting conditions without stimulation. C Normalized YPet/ECFP FRET ratio of BKTS and RKTS over time at 300 s in the no stimulation conditions. BKTS, n = 8; RKTS, n = 7. D, E Verification of biosensors sensitivity and reversibility. D YPet/ECFP FRET ratio images of BKTS and RKTS biosensors at 0 s, 60 s, and 300 s under brief electrical stimulation. E Normalized YPet/ECFP FRET ratio curves of BKTS and RKTS before and after a single suprathreshold stimulation, using the FRET ratio at the last moment before stimulation as the baseline. BKTS, n = 6; RKTS, n = 7. The red arrow indicated the time point when the stimulation was applied. F, G Verification of biosensor linearity. F YPet/ECFP FRET ratio images of SH-SY5Y cells transfected with BKTS and RKTS biosensors before and after 5 min of electrical stimulation at 2 Hz, 10 Hz, and 20 Hz. G Linear regression analysis and statistical comparison of normalized YPet/ECFP FRET ratios for BKTS and RKTS at 300 s (Green line, fitting curve using the least squares fit). BKTS (2 Hz, n = 7; 10 Hz, n = 9; 20 Hz, n = 7); RKTS (2 Hz, n = 11; 10 Hz, n = 11; 20 Hz, n = 7). One-way ANOVA. ***P < 0.001. Data are mean ± SEM.
