Swab samples, a noninvasive method for skin surface evaluation, of 29 subjects were collected on both cheeks on D0 and D28 and in D84 only on the side of the face treated with M89PF. After the zone of interest and size were defined, a first swab previously dampened in the cocktail solution was rubbed onto the surface of interest for 45 seconds. Then, the same step was performed with a second swab previously dampened in the cocktail solution. Both swabs were introduced into a sample tube and stored at −20°C.
For sample extraction, each cotton bud was removed from its stick and placed into another 0.5mL sample tube drilled at the bottom itself and introduced into a 2 mL sample tube. The content was centrifuged for 3 min at 18,000 g at 4°C. The liquid obtained was collected for biochemical exploration.
The IL-8, IL-1alpha and IL1Ra assays were performed by Elisa (R&D Systems Inc., Minneapolis, MN, USA). A plate with a monoclonal antibody respectively specific for IL-8, IL-1α or IL1RA was prepared. Then, the sample of interest was deposited in order to bind to the antibody and to add a polyclonal antibody recognizing the antigen. A secondary antibody bound to the enzyme was added to convert it into a detectable entity (450 nm) directly proportional to the amount of IL-8, IL-1α or IL1RA.
The PGE2 assay, to test the COX-2 (Cyclo-oxygenase-2) activity, was carried out by Elisa (R&D Systems Inc., Minneapolis, MN, USA). A plate with anti-mouse IgG antibodies attached was prepared. A mixture of labeled antigens (alkaline phosphatase conjugated-PGE2) and antigens to be assayed (sample) were deposited on the plate. The Prostaglandin E2 antibody and substrate were then added and the enzymatic reactivity converted to a detectable entity (450 nm) inversely proportional to the amount of PGE2.
The Catalase activity was measured by enzyme kit (R&D Systems Inc., Minneapolis, MN, USA).
This method consists of measuring the residual content of hydrogen peroxide (H2O2) following the catalase action, which induced the fluorescence of resorufin. This is an indirect assay of hydrogen peroxide. The resorufin intensity is inversely proportional to the activity of catalase.
SOD activity was tested by enzyme kit (R&D Systems Inc., Minneapolis, MN, USA).
SOD catalyzes the conversion reaction of O2- into hydrogen peroxide and oxygen. These superoxide ions are produced by the conversion of xanthine to hydrogen peroxide and catalyze the WST-1 conversion reaction to produce a coloration detected at 450 nm.
For sample extraction, each cotton bud was removed from its stick and placed into another 0.5mL sample tube drilled at the bottom itself and introduced into a 2 mL sample tube. The content was centrifuged for 3 min at 18,000 g at 4°C. The liquid obtained was collected for biochemical exploration.
The IL-8, IL-1alpha and IL1Ra assays were performed by Elisa (R&D Systems Inc., Minneapolis, MN, USA). A plate with a monoclonal antibody respectively specific for IL-8, IL-1α or IL1RA was prepared. Then, the sample of interest was deposited in order to bind to the antibody and to add a polyclonal antibody recognizing the antigen. A secondary antibody bound to the enzyme was added to convert it into a detectable entity (450 nm) directly proportional to the amount of IL-8, IL-1α or IL1RA.
The PGE2 assay, to test the COX-2 (Cyclo-oxygenase-2) activity, was carried out by Elisa (R&D Systems Inc., Minneapolis, MN, USA). A plate with anti-mouse IgG antibodies attached was prepared. A mixture of labeled antigens (alkaline phosphatase conjugated-PGE2) and antigens to be assayed (sample) were deposited on the plate. The Prostaglandin E2 antibody and substrate were then added and the enzymatic reactivity converted to a detectable entity (450 nm) inversely proportional to the amount of PGE2.
The Catalase activity was measured by enzyme kit (R&D Systems Inc., Minneapolis, MN, USA).
This method consists of measuring the residual content of hydrogen peroxide (H2O2) following the catalase action, which induced the fluorescence of resorufin. This is an indirect assay of hydrogen peroxide. The resorufin intensity is inversely proportional to the activity of catalase.
SOD activity was tested by enzyme kit (R&D Systems Inc., Minneapolis, MN, USA).
SOD catalyzes the conversion reaction of O2- into hydrogen peroxide and oxygen. These superoxide ions are produced by the conversion of xanthine to hydrogen peroxide and catalyze the WST-1 conversion reaction to produce a coloration detected at 450 nm.
