NeoLux; Viability assessment by loss of efflux protein function

The patented NeoLUX technology is a life cell test that measures the state of homeostasis or cell damage by fluorescence readout. The technology has been optimized for high throughput on 96- and 384-well plates, suitable for commercial fluorescence readers according to a very simple protocol limited to the addition of the fluorescent marker in the culture medium and two fluorescent measurements.
The NeoLUX technology is based on photo-induction of a biosensor leading to a fluorescence signal. This fluorescence increases is only observed on cells in homeostasis prior to biosensor addition. Calculation of the fluorescence ratios before and after LED application leads to an accurate and dose-dependent measurement of the state of cellular damage that follows chemical or physical toxicity induced by the sample.

NeoLUX test Method: Principle

The experimental procedure is simple. The TO biosensor is added to the cell culture medium and becomes fluorescent after association with the cell nucleic acids. When TO is photoactivated by LED illumination at 480 nm, it’s relaxation is accompanied by an energy transfer to the intracellular dioxygen molecule (³O₂) resulting in the production of singlet oxygen (¹O₂) and a cascade of Reactive Oxygen Species (ROS) production including superoxide anion (O₂^.-) and hydroxyl radical (OH^.) (see fig. 1). The antioxidant effect of a substance can then be measured by its ability to counteract this intracellular ROS production.

Dose-Effect curves 

Intracellular antioxidant effect is revealed by a time delay in the fluorescence increase triggered by the LED-dependent photo-activation of TO. Figure 2 illustrates the fluorescence profiles of human HepG2 cells treated 4 h at different resveratrol concentrations. Resveratrol is a powerful cellular antioxidant that is known for being active both as a ROS scavenger and as an inducer of the cell natural defense driven by Nrf2 pathway. Control (non-treated) curve is illustrated in black. Differences between control and sample Area Under Curves (AUC) allow to establishing an index that qualify the intracellular antioxidant effect of the sample.

EC₅₀s and other parameters revealing antioxidant effects

Calculation of NeoLUX index at different sample concentrations allows to establish dose-effect profiles that fits well with sigmoid regression, allowing the evaluation of the EC₁₀ (Efficacy Concentration 10%, threshold of cellular effect), the standard EC₅₀ (Efficacy Concentration 50%) and the EC₉₀ (Efficacy Concentration 90%, weakest dose at which the compound acts with a maximum effect). Figure 3 illustrates an example of dose-effect curves obtained after treatment with resveratrol for 1 and 4 h on human HepG2 cells.

NeoLUX : a multifaceted test method

More generally, NeoLUX fluorescence profiles provide information on several parameters that are specific to the tested sample and the chosen cell model. In addition to the antioxidant effect described above, NeoLUX test highlights pro-oxidant effects (left-shift of the sample profile) and cytotoxic effects (initial fluorescence is high and illumination remains non effective) (Figure 4), bringing added value to the method. It is also noteworthy that the NeoLUX technology works on any kind of eukaryotic (animal, vegetal, fungal) and prokaryotic (bacteria) cell models.

Features

• For 1000 or 4000 mesure points in 96-well plates
• One-Step procedure
• No wash
• Standard procedure to most cell lines ( optimisation kit available if needed)
• Adapted to cell lines, hiPSCs,primary cell,...
• 1 solution in the kit ( biosensor to be diluted in the culture medium)
• Storage 4°C
• Time to exiration: 6 months
• Also available for 384-w plates.

Mechanism

The process is called light-up cell system because the fluorescence level of the fluorescent biosensor increases during its photoinduction by illumination. The biosensor passively enters the cells but is quickly removed from functional cells by efflux transport proteins, resulting in a low fluorescent signal. When the light is applied, biosensor photoinduction generates intracellular ROSs, which alter the cell homeostasis or cell’s ability to release the biosensor, triggering its massive entry within the cells, and resulting in an increased fluorescence signal. If cells have been previously incubated with a toxic substance, causing alteration of efflux or other cellular functions, the biosensor enters massively, leading to a high fluorescence signal with no increase after light application.

 Safety

This product is for research purposes only and not for human or therapeutic use. Potentially harmful. Avoid prolonged or repeated exposure. Avoid getting in eyes, on skin, or on clothing. Wash thoroughly after handling. If eye or skin contact occurs, wash affected areas with plenty of water for 15 minutes and seek medical advice. In case of inhaling or swallowing, move individual to fresh air and seek medical advice immediately.

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