LUCS (Light-Up Cell System), a universal high throughput assay for homeostasis evaluation in live cells

Abstract

Observations of fluorescent cyanine dye behavior under illumination at 500 nm lead to a novel concept in cell biology allowing the development of a new live cell assay called LUCS, for Light-Up Cell System, measuring homeostasis in live cells. Optimization of the LUCS process resulted in a standardized, straightforward and high throughput assay with applications in toxicity assessment. The mechanisms of the LUCS process were investigated. Electron Paramagnetic Resonance experiments showed that the singlet oxygen and hydroxyl radical are involved downstream of the light effect, presumably leading to deleterious oxidative stress that massively opens access of the dye to its intracellular target. Reversible modulation of LUCS by both verapamil and proton availability indicated that plasma membrane proton/cation antiporters, possibly of the MATE drug efflux transport family, are involved. A mechanistic model is presented. Our data show that intracellular oxidation can be controlled by tuning light energy, opening applications in regulatory purposes, anti-oxidant research, chemotherapy efficacy and dynamic phototherapy strategies.

Figure 1

LUCS assay demonstration and optimization on HepG2 cells: (A) light-induced fluorescence observed in live cells treated with seven different cell fluorescent dyes (1 h, 4 µM), then illuminated full power with a Varioskan reader up to 900 s; (B–D) LUCS experiments using thiazole orange (TO): (B) TO (1 h, 0.25–32 µM) dose-dependent evolution of LUCS fluorescence ratios (F_post/F_pre) with F_post measured 1 (dark circles) or 60 min (open circles) after light application; (C) fluorescence curves obtained after treatment (24 h) at increasing doses of chloroquine, T = 0: addition of TO 4 µM, T = 40 min, LED illumination (470 nm, 240 mJ/cm²); (D) chloroquine dose-response curve as measured by LUCS fluorescence ratios. Data presented are representative of 2 (A,B) and 6 (C,D) experiments.

Figure 2

LUCS as a cytotoxicity assay. Regression analysis of 3T3 NRU vs LUCS Log EC₅₀s obtained from dose-response experiments using 53 out of the 97 substances of the ACuteTox European Program databank. Monographs of each dose-response are presented as Supplementary data, see Figure S1.

Figure 3

Investigation on pre-illumination stage in HepG2 cells. (A) TO dose-response steady state experiments (150 min post TO-labelling) in healthy (dark circles) and chloroquine-treated (1 mM, 24 h, open circles) cells; insert: linear regression analysis based on the six lowest TO concentrations; (B,C) effect of efflux modulators on fluorescence levels in healthy cells preloaded with TO (B) or SYTO-13 (C) (2 µM); (D) verapamil (200 µM) was added (step 1, T = 30 min post TO 0.5 µM addition, black and open circles), removed (step 2, open circles) and re-added (step 3, open circles); (E) cells treated with TO 8 µM were maintained at pH 7, then at various pHs from 5.5 to 9; (F) cells treated with TO 0.5 µM were maintained at pH 9.5 (black circles) or at pH 9.5 and pH 7 (open circles), then submitted to 600 µM verapamil. Data presented are representative of 2 (A), 3 (B,C,E,F) or 4 (D) experiments.

Figure 4

Investigation on post-illumination stage, involvement of reactive oxygen species (ROSs): (A–C) CHO cells were preloaded with 4 µM TO for 20 min at room temperature, DMPO (50 mM) was added, LED light (240 mJ/cm²) was applied (B,C) or not (A) and electronic paramagnetic resonance (EPR) was recorded in the following minute; in (C) H₂O was replaced by D₂O 20 min before TO was added; (D) kinetic RPE follow-up including two successive light applications showing the amplitude of the largest RPE peak (3496 ± 2 gauss).

Figure 5

“Trojan horse” mechanistic model of LUCS: 1, TO is mainly removed out of the cell by efflux transport limiting its access to nucleic acid targets; low fluorescence (F_pre) is observed; 2, light is applied inducing ROS production that alters efflux and/or other cell functions; 3, massive entry of TO triggers increase in fluorescence emission (F_post).