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. Author manuscript; available in PMC: 2026 May 17.
Published in final edited form as: ACS Sens. 2026 Feb 6;11(2):909–922. doi: 10.1021/acssensors.5c01707

Figure 3.

Figure 3.

Multiplexed MDF biosensors implemented in 3D neuro-glial spheroids (NGS) resolves cell-type specific FRET. Control MDF biosensors were expressed in a series of SHSY5Y/HMC3 spheroids. Green: S0 = naive SHSY5Y; S1 = mNg; S2 = ShY-L-mNg; Red: H0 = naïve HMC3; H1 = mScI3; H2 = ShR-L-mScI3. (A–D) Live-cell fluorescent imaging of two distinct S1+H1 NGS with green SHSY5Y derived neurons and red HMC3 microglia. Transmitted light overlays in panel A, and panel C highlights NGS perimeter. Cell nuclei are stained with Hoechst dye (blue). (E) Comparison of SHSY5Y FLT in NGS spanning all HMC3 biosensor conditions. (F) No significant difference between SHSY5Y donor only (S1) across spheroid type. (G) No significant difference between SHSY5Y donor + acceptor (S2) across all NGS compositions. (H) Resulting green channel FRET is not changed across all NGS compositions. (I) Comparison of HMC3 FLT in spheroids spanning all SHSY5Y biosensor conditions. (J) No significant difference between HMC3 donor only (H1) across NGS. (K) No significant difference between HMC3 donor + acceptor (H2) across all spheroid compositions. (L) Resulting red-channel FRET is not changed across all NGS compositions. Data represent mean ± SD of n = 3 biological replicates; significance determined by one-way ANOVA (ns = p > 0.05; **** = p < 0.0001).