SKIN PROTECTION - Oxidative Stress protection

COSMETIC TESTING

Fluofarma offers a panel of validated cell-based assays allowing the quantification of key phenotypic and molecular events at the single-cell level and enabling to evaluate the potential benefits of compounds. Most of the cellular assays listed below can be performed on our automated screening platforms. Assays can be adapted to a different cell model, or fully developed, according to your need.

SKIN PROTECTION - Oxidative Stress protection

Skin is a major target for toxic insult by a broad spectrum of physical and chemical agents that are capable of altering its structure and function. These environmental toxins or their metabolites are inherent oxidants and/or directly or indirectly drive the production of a variety of reactive oxidants also known as reactive oxygen species. The “antioxidant power” of a product is an expression of its capability both to defend against the action of the free radicals and to prevent degenerative disorders.

FLUOFARMA offers many in vitro models to evaluate anti-oxidant properties of ingredients or products :

- Cell viability after oxidative stress e.g. cytolysis or mitochondrial potential (NHEK, HaCaT)
- Nrf-2 pathway for oxidative stress protection
- Inflammatory cytokine release
- Lipid peroxidation measurement
- Reactive Oxygen Species (ROS) measurement

Antioxidant properties in keratinocyte – ROS measurement

Antioxidant properties of ingredients can be addressed in primary human keratinocyte models through ROS measurement. NHEK (Human primary keratinocytes) cells were plated in 96-well plates. After 24h of culture, cells were treated with tBHP, Menadione or Cumene hydroperoxyde (ROS inducers) with or without N-Acetyl-Cysteine or Vitamin E (antioxidant reference compounds). We performed a Flow cytometry measurement of ROS production in-cellulo.

Treatment with oxidative compound led to a strong ROS production. N-Acetyl-Cysteine and vitamin E protected cells against the oxidative stress produced by diminishing ROS levels.

Lipid peroxidation measurement in primary keratinocytes

Antioxidant properties of ingredients can be addressed in primary human keratinocyte models through lipid peroxidation measurement. NHEK (Human primary keratinocytes) cells were plated in 96-well plates. After 24h of culture, cells were treated with tBHP (peroxidation agent) with or without N-Acetyl-Cysteine or Vitamin E (antioxidant reference compounds). We performed a Flow cytometry measurement of lipid peroxidation in-cellulo.

Treatment with oxidative compound led to a strong peroxidation production. N-Acetyl-Cysteine and vitamin E protected cells against the oxidative stress produced by diminishing lipid peroxidation levels.

Antioxidant properties in keratinocytes - Mitochondrial Potential

Normal Human Epidermal Keratinocytes (NHEK) were seeded in 96-well plates. After 24h of culture, cells were treated with N-Acetyl-Cysteine 10mM (antioxidant reference compound) for 24h and then treated with Hydrogen Peroxide (H2O2) in the presence or not of NAC (for 4h). The protective effect of compounds against oxidative stress was evaluated by measuring mitochondrial membrane potential with a fluorescent cell-permeant cationic mitochondrial probe (accumulated in mitochondria, depending on membrane potential). Treatment with H2O2 led to a strong mitochondrial depolarization with a dose-dependent effect. N-Acetyl-Cysteine protected cells against the oxidative stress produced by H2O2..

Antioxidant properties in keratinocytes - Cytolysis kinetics

Protective properties of ingredients can be addressed in keratinocyte models of H2O2 -induced oxidative damage through cytolysis or mitochondrial toxicity measurements. ROS production can also be assessed.

HaCaT (Human immortalized keratinocytes) cells were plated in 96-well plates. After 24h of culture, cells were treated with H2O2 with or without N-Acetyl-Cysteine (antioxidant reference compound). We performed a kinetic monitoring of H2O2 -induced cytolysis and its inhibition by N-Acetyl-L-Cysteine (NAC).

Treatment with H2O2 led to a strong cytolysis. N-Acetyl-Cysteine completely protected cells against the oxidative stress produced by H2O2 with levels of cytolysis equal to the untreated condition.