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At VivaCell, we have adapted specific protocols “in vitro” to study the anti-tumoural activity of phytomedicines and nutraceuticals. More than 100 parameters including cellular, molecular and biochemical techniques are offered to our client, and by using such parameters we run projects adapted to each phytodrug and tailored to customer’s needs.
In vitro testing methods include proliferation inhibition assays using human tumour cell lines, as well as clonogenic assays with human tumor xenografts, human tumor cell lines, and hematopoetic stem cells. More than 350 solid tumor models have been established in serial passage as well as 20 permanent tumor cell lines (co-operation). In addition, a panel of 60 cell lines, commonly used in cancer research such as A549, MCF-7, Caco-2, HT29 or PC3, are also available. Cancer cell lines overexpressing the MDR1 gene are also available for drug research.
In vivo assays include subcutaneous models, orthotopic models with human xenografts, murine tumour models (co-operation) and models of topic co-carcinogenesis inducing papilloma-like skin tumours in athymic mice.
Angiogenesis(e.g. VEGF, angiopoietin I and II, nephrin, i.e.). In vitro3D assays (spheroids), HUVECs and Matrigel Basement Membrane Matrix, In vivocorneal neovascularization assays, 3D assays (spheroids injected s.c.),
Parameters:
· Proliferation, XTT assays, determination of DNA content by FACS analyses
· Cytokines (e.g. IL-6), growth factors (e.g. EGF) etc.
· Cell cycle analyses, cytotoxicity by measuring PI staining
· Determination of DNA fragmentation (apoptosis) by TUNEL
· Transmembrane potential of the mitochondria, calcium and pH mobilisation
· Reactive oxygen species (ROS)
· Fas clustering and signalling in type I and II cells for apoptosis
Caspases (3, 7, 8, 6 and 9) activity by spectrofluorometry and western blots
· Bcl-2, Bcl-X, Bax, Bid and cytoplasmic cytochrome C by western blots and PCR.
· Apoptosis and cell cycle analyses in transfected MDR (pgp-1) cell lines
· Phosphatidyl serine expression at the cell surface by Anexin V binding.
· Integrity of tubulin and actin by fluorescence microscopy
· In vitro assays for tubulin polimerization.
· Cyclin activities by IP and Western blots
· Activation of Transcription Factors
· Signalling studies MAPKs, IKK, ie..
· Determination of Telomerase activity in HeLa cells by Telomerase PCR ELISA
· Customized “knock out” cell lines for specific genes
· DNA microarrays for cancer-related genes in tumoural cell lines
Other specific cellular and molecular techniques are available upon request.
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