Extended Data Fig. 2: Further in vitro characterization of nLightR2 and nLightG2.
From: Next-generation multicolor indicators for in vivo imaging of norepinephrine
a, Fluorescence response (mean ± s.e.m.) of nLightR2 to HBSS or HBSS supplemented with neurotransmitters (10 µM). Data from six (NE) or three (others) independent experiments with n = 42 (NE) or n = 21 (others) cells. One-way Welch ANOVA with Dunnett’s T3 test: NE, p < 10−¹⁴; Epi, p < 10−14; DA, p < 10−14; 5-HT, p = 0.999; GABA, p = 0.779; His, p = 0.955; Glu, p = 0.998; Ach, p = 0.907; Ado, p = 0.681. b, Same as in (a) for nLightG2. Data from three independent experiments with n = 21 cells per ligand. Welch ANOVA with Dunnett’s test: NE, p < 10−14; Epi, p < 10−14; DA, p < 10−14; 5-HT, p < 10−14; GABA, p = 0.971; His, p = 0.976; Glu, p = 0.527; Ach, p = 0.994; Ado, p = 0.999. c, Fluorescence dose–response of nLightR2 (stably expressed in HEK293T cells) to NE and DA. Curves fitted with four-parameter models to determine EC₅₀ values (n = 3 wells per point; mean ± s.e.m.). d, Same as in (c) for nLightG2. e, Same as in (c) for GRABNE2m. f, One-photon excitation and emission spectra of nLightR2 (transiently expressed in HEK293T cells) in unbound (dashed) and NE-bound (10 µM, solid) states. Data from four independent experiments (n = 4 wells total). g, Same as in (f) for nLightG2 (n = 3 wells, three experiments). h, Two-photon excitation spectra of nLightR2 in HEK293T cells. Spectra normalized to Apo form at 1020 nm; ratio between saturated (Sat) and Apo shown as dotted line (n = 3 dishes). i, Same as in h for nLightG2, normalized to Apo form at 950 nm. All experiments repeated at least three times with consistent results.
