Fig. 1. Generation and validation of ferritin binding nanobodies for magnet stimulation.

Serial dilutions of nanobodies or BSA (100ul) were incubated on plates coated with human spleen ferritin (1ug/ml) and binding quantified by ELISA after incubation with anti-HA-HRP antibody. A. Quantification of anti-ferritin nanobodies from CDR3 groups 6 and 26 binding to human spleen ferritin. B. Oscillating magnetic field treatment (465kHz, 30mT) significantly increases calcium-dependent SEAP release from 293T cells transfected with Nb-GFP-TRPV1^Ca2+, Nb-Ft-2-TRPV1^Ca2+ and Nb-Ft-14-TRPV1^Ca2+ and GFP-mFerritin. Data analyzed by two-tailed, unpaired t-test with Welch’s correction. Nb-GFP-TRPV1^Ca2+, basal vs. RF * p = 0.04, n = 8 & 8; Nb-Ft-2-TRPV1^Ca2+, basal vs. RF * p = 0.02, n = 7 & 8; Nb-Ft-14-TRPV1^Ca2+, basal vs. RF * p = 0.04, n = 7 & 8. C. Normalized Fluo-4 fluorescence (ΔF/F₀) in Neuro2A cells expressing Nb-Ft-2-TRPV1^Ca2+ with (786 cells) or without (1659 cells) magnet treatment. Data were analyzed by two-way ANOVA with Tukey’s multiple comparison test, **** p < 0.0001. D. Cumulative change in ΔF/F₀ of Neuro2A cells expressing Nb-Ft-2-TRPV1^Ca2+ with (786 cells) or without (1659 cells) magnet treatment. Data were analyzed by Mann Whitney U test **** p < 0.0001. E. Changes in RCaMP fluorescence normalized to baseline fluorescence (ΔF/F0) with magnet treatment of HEK-293T cells expressing RCaMP alone (54 cells), TRPV1^Ca2+ (107 cells) or Nb-Ft-2-TRPV1^Ca2+ (101 cells). Error bars represent mean +/−SEM. F. Cumulative change in ΔF/F₀ with magnet treatment of HEK-293T cells expressing RCaMP alone (54 cells), TRPV1^Ca2+ (107 cells) or Nb-Ft-2-TRPV1^Ca2+ (101 cells). AUC was calculated for the period of magnet exposure between 48–180 seconds (after focus adjustment) and analyzed by ordinary one-way ANOVA with Tukey’s multiple comparison test, **** p < 0.0001. Data are shown as mean ± SD.