Fig. 1

A Relative fluorescence of GECI’s GCaMP6f⁷⁹ and RCEPIA³⁶/RGEDI across Ca²⁺ concentrations present in the cytosol, endoplasmic reticulum, and extracellular milieu modeled from previously reported Hill coefficients and K_d values. B Representative fluorescence images of rat primary cortical neurons transfected with GCaMP6f or RGEDI at baseline, during 30 Hz × 3 s field stimulation, or after 2% NaN₃ treatment, scale = 10 μm. The experiment was repeated six times for each condition with similar results. C, D Representative trace of the time course of standardized ΔF/F fluorescence after 30 Hz × 3 s stimulation (open arrowhead), and after NaN₃ treatment (black arrowhead) of GCaMP6f (C) and RGEDI (D) expressing neurons. E To determine whether RGEDI signals were specific for cell death and not confounded by physiological Ca²⁺ transients, the ratio of the maximum signal from electrical stimulation to toxin treatment per neuron are shown, demonstrating that GEDI does not respond to physiological Ca²⁺ transients (n = 6 cells/group, 1 cell per coverslip, *** Unpaired, two-tailed T-test, p < 0.0001). Error bars represent SEM. F Design of RGEDI-P2a-EGFP cassette for pseudo-ratiometric expression in neurons. G Illustration of color change in red:green image overlay expected in a live versus dead neuron. Live neurons have EGFP (green) and basal RGEDI (red) fluorescence (overlaid as yellow) within the soma of the neuron, surrounded by green fluorescence that expands through the neurites. Dead neurons display yellow fluorescence throughout, with edges of red fluorescence around the soma and throughout degenerating neurites as extracellular Ca²⁺ permeates the membrane (arrows). H Time course images of rat primary cortical neurons expressing RGEDI-P2a-EGFP at 24–39 h post transfection (hpt). Neuron 1 shows characteristic morphology features of life through 27 hpt at which time it also shows elevated GEDI ratio (yellow asterisk), followed by loss of fluorescence at 39 hpt. Neuron 2 remains alive through time course, scale = 20 μm. Color scales are annotated in arbitrary units for each color channel. I Quantification of change in GEDI ratio of Neurons 1 and 2 in (H). J Quantification of GEDI ratio in rat primary cortical neurons before (blue dots) and from a separate well 5 min after NaN₃-induced neuronal death (green dots = live, red dots = dead). The Black dotted line represents the calculated GEDI threshold. (*** One-tailed T-test p < 0.0001). K Quantification and classification of death in neuronal cultures at 24 and 48 h after co-transfection of RGEDI-P2a-EGFP and an N-terminal exon 1 fragment of Huntingtin, the protein that causes Huntington’s disease, with a disease-associated expansion of the polyglutamine stretch (HttEx1Q97) to induce neuronal death using the derived GEDI threshold from (J) to define death. Independently, neurons were scored as dead (red), or live (green) by eye using EGFP at 24, 48, and 72 post transfection. Neurons that were classified as live by eye but above the GEDI threshold and classified as dead at the subsequent time point were called human errors (black).