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SkinAxis evaluates changes in age-related skin markers. Collagen and elastin expressions are evaluated by PCR and immunoblot. Expression of growth factors and cytokines are detected by ELISA, metabolism by luminescence assay, and antioxidant enzymes by colorimetric assays or immunostaining.
SkinAxis evaluates the formation of functional barrier as a result of differentiation using protein or RNA expression of specific differentiation markers in 2D or 3D skin models by PCR, immunoblot, or immunohistochemical staining.
Pigmentation and Brightening
SkinAxis measures melanin synthesis as the total amount of melanin produced in response to the treatment with active ingredient in 2D or 3D skin models using a colorimetric assay. SkinAxis also evaluates tyrosinase expression and activity after treatment with active ingredients for whitening or tanning application using colorimetric assay.
SkinAxis evaluates expression of specific differentiation markers in 2D or 3D models,
inflammatory cytokine release, cell viability and metabolism using the MTT test, ATP content by luminescence assay, and detoxification enzymes by ELISA.
SkinAxis evaluates collagen production in human dermal fibroblasts, MMP activity using
colorimetric assays, and MMP expression using PCR or ELISA.
UV Protection and DNA damage Assay
SkinAxis evaluates morphological changes after UVA or UVB exposure by H&E staining of skin equivalents and DNA damage by immohistochemical staining. Highly sensitive quantitative measurement of DNA damage by ELISA to determine cyclobutane pyrimidine dimers (CPD)
SkinAxis evaluates enzymes of cell antioxidant defense system by colorimetric assays or
immunostaining, ROS formation by antioxidant luminescence assay, and AGE formation following glycation by immunofluorescence or immunoblot.
Skin Irritation Assay
Skin irritation assay is used to determine reversible damage to the skin by application of a test compound to the reconstructed human epidermis. Compound exposure that induces skin irritation evaluated by release of inflammatory mediators such as the cytokine Interleukin 1 alpha (IL-1a) or MTT assay.
QuantiSig Skin Technology
The measurement of different biological responses of the skin is of great importance in the activity and toxicity testing of molecules employed in skin product development. Various methods have been developed to assess the effects of compounds on the various skin features. These methods rely mainly on the measurement of changes in the expression of RNA or protein markers within treated skin cellular components (e.g. keratinocytes, dermal fibroblasts, melanocytes). Different markers are assessed as indicative endpoints of specific skin responses using mostly immunocytological and SkinAxis, LLC immunohistological approaches. These methodologies are offer often limited sensitivity, and are highly variable and difficult to objectively quantify. To circumvent these limitations, SkinAxis has developed the QuantiSig technology that allows the analysis of the activation of different pathways representative of specific biological responses of the skin in a reproducible, highly sensitive, and measurable fashion. QuantiSig is a cell engineering technology that can be applied both in 2D cell cultures as well as in the reconstitution of 3- dimensional skin models without interfering with the normal physiological responses of the cells.
QuantiSig allows the different cell types composing the skin tissue to stably carry a reporter gene whose expression is controlled by genetic elements activated during the stimulation of various pathways. The activation of the pathway following the application/exposure to chemical and biological compounds can then be monitored enzymatically in the engineered cells. Using different reporter genes, different cells and/or pathways can be evaluated at the same time. The QuantiSig technology can be used to monitor the following cellular pathways in 2D cultures and reconstituted 3D tissues:
DNA damage response
QuantiSig-DDR provides a very sensitive measurement of the DNA damage response pathway. The test can be applied to determine the effects of UV irradiation, sun protection applications on the DNA damage response pathway in skin as well as anti-cancer drug testing affecting the p53 pathway.
QuantiSig-AOR allows to test the anti-oxidative response pathway involved in the modulation of the Nuclear factor erythroid-related factor 2 (Nrf2) and the expression of cytoprotective genes. Constitutive activation of Nrf2 has been found in many cancers, resulting in resistance against chemotherapy and radiotherapy in cancer cells. Thus, screening for Nrf2 inducers and inhibitors is important to develop therapies for stress-induced diseases including cancer, and for the identification of anti-oxidant components for skin care products.
PPARs (PPARα, PPARβ/δ, and PPARγ) pathway
QuantiSig-PPAR identifies the activation of Peroxisome Proliferator-Activated Receptors (PPARs), members of the nuclear receptor family of ligand-activated transcription factors. The system detects all three-member subfamily PPARα, PPARβ/δ, and PPARγ, which control the expression of genes involved in lipid and carbohydrate metabolism, vascular biology, tissue repair, cell proliferation and differentiation. Testing of PPAR agonists is suitable for drug discovery applications for various conditions (e.g. diabetes, coronary artery disease, obesity, and cancer) and the identification of agents supporting cell regeneration.
XRE- xenobiotic responsive element (XRE)
QuantiSig-XRE allows testing the activity of the aryl
hydrocarbon receptor (AhR). AhR is a cytosolic ligand-activated transcription factor that directly interacts with drugs metabolites and environmental toxins collectively known as xenobiotics. This receptor is widely distributed in vertebrates and is expressed in skin cells, in which exogenous and endogenous ligands are abundant, where it is involved in the metabolism of xenobiotics as well as in the regulation of skin pigmentation and skin inflammation.
Human macrophages differentiated from Peripheral Blood Mononuclear Cells (PBMC)-derived monocytes. For more information visit: http://www.skinaxis.com
Sample Collection and Processing
CD14+ monocytes were isolated from buffy coats of normal human volunteers by positive selection using an immunomagnetic cell separation method. Monocytes were differentiated into macrophages by culturing cells for 10-13 days in the SkinAxis proprietary medium.
The purity of PBMC-derived CD14+ monocytes, as measured by flow cytometric analysis, is ≥95%. Macrophages are >99% positive for the pan-macrophage CD68 marker and show efficient phagocytic function.
Stability and Storage
Macrophages are cryopreserved in the SkinAxis Freezing Medium and are stable for long-term storage at -152oC or in liquid nitrogen. Storage at -80oC is NOT recommended. Once thawed, the cells must be used immediately.
Cells are derived from donors with undetectable HIV-1, hepatitis B and hepatitis C. Macrophages are characterized by morphological analysis, specific marker expression, and phagocytic ability.
Primary cells from human blood are a potential biohazard. Treat as potentially infectious. All human sourced products should be handled at the Biological Safety Level 2 to minimize exposure of potentially infectious products.