NeoStress

Neostress antioxidant live cell assay was developed from the Light Up Cell System technology that allows for fine monitoring of intracellular ROS production. The technology has been optimized for high throughput on 96- and 384- well plates, suitable for commercial fluorescence readers according to a simple protocol limited to the addition of the Solution A1 in the culture medium and twenty runs of illumination/fluorescence measurements.

Mechanism

Neostress is based on the activation of an intracellular photosensitizer in a protocol that only requires a succession of light flashes and fluorescence readings. The process is called light-up cell system because the fluorescence level of the 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. The increase in fluorescence is delayed or abolished in cells previously incubated with an antioxidant substance acting by neutralizing the free radicals produced by the cells under illumination.

Features:  

• For 400 measure points in 96-well plates
• One-step procedure
• No washes
• Storage 4°C
• Time to expiration: 6 months after receipt
• Standard procedure to most primary cells, hiPSCs, immortalized cell lines,… (optimization kit available if needed)
• Can be used on multiplexing

General Assay Protocol

1. Prepare samples 10X concentrated
2. For positive control condition, add 63μL of culture medium without serum in Solution B tube. Mix with the pipette
3. Remove culture medium from cells, then add 90μL of culture medium without serum
4. Add 10μL of positive control and sample conditions to wells
5. Incubate for 1h in incubator
6. For 96 wells, prepare 3.13μL of Solution A1 + 2596.9μL of culture medium without serum. Mix with the pipette
7. Add 25μL of this diluted Solution A1 per well. Avoid exposing to excessive light during this step and next steps.
8. Incubate plate at determined time during optimization protocole at room temperature
9. Read fluorescence (Fpre) at the following wavelengths:
10. λExcitation= 505nm (±10nm)
11. λEmission= 535nm (±10nm)
12. Illuminate the plate using the AOP illuminator (AOPLight B1 or AOPLight HTS)
13. Read fluorescence (Fpost)
14. Repeat steps 10 and 11 twenty times

N.B.: Non-specific fluorescence can be measured by reading fluorescence before step 6.

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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