SANIPEDIA Research Lab

Transcription

1 SANIPEDIA Research Lab Cells and In Vitro Assays Catalog

2 Index A. CELL PORTFOLIO 3 B. GENERAL ASSAYS 4 B.1 WHOLE CELL ASSAYS 4 B.1.1. Viability and Toxicity 4 B.1.2. Proliferation 4 B.1.3. Apoptosis 4 B.2. PROTEINS AND GENES 5 B.2.1. Protein Purification and Analysis 5 B.2.2. Molecular Biology 5 B.2.3. Cell Staining 5 B. 3. FUNCTIONAL ASSAYS 5 C. SPECIALIZED ASSAYS 6 C1. SPECIALIZED NEUROLOGIC ASSAY AND MODELS 6 C2. BLOOD BRAIN BARRIER MODEL 6 C3. METABOLIC MODEL 7 D. CUSTOMIZED PLATFORMS 8 D.1 8 D.2 10 D

3 A. Cell Portfolio Cell Type Species Cell format mrna Rat Mouse Human Cryovials Homog. extracts Primary cortical astrocytes * * * * * * Fixed cells Brain Primary hippocampal astrocytes * * * * * * Primary cortical neurons * * * * * * Cancer Primary hippocampal neurons * * * * * * Primary microglia * * * Glioma * * * * * Glioblastoma * * * * * Neuroblastoma * * * * * Breast Cancer * * * * * Immune system Monocytes * * * * * Eye Retinal Ganglion cells * * * * * Stem cells Mesenchymal stem cells Adipose Tissue-derived MSC * * * * * * * Bone Marrow-derived MSC * * * * * * * Neural stem cells * * * * * * * 3

4 B. General Assays B.1 Whole Cell Assays B.1.1. Viability and Toxicity Evaluation of cell s death Evaluation of cell s vitality-metabolism Nuclear Staining: Trypan blue/ P.I/ Hoechst/ YO-PRO Cellular substance release: LDH assay Bioluminescent assay (ATP based luciferase reaction) Esterase substrate (i.e. Calcein-AM) HCS assay mitochondrial damage MTT assay Glucose Uptake Measurement of oxidation/reduction state B.1.2. Proliferation Evaluation of cell s clonigenic performance Evaluation of cell s DNA syntesis Evaluation of cell s metabolic activity Membrane staining (crystal violet, alamar blue, ecc) Fluorescent-based assay BrdU staining Fluorescence/luminescence assay XTT assay Fluorescence/luminescence assay (i.e BodiPy dutp) B.1.3. Apoptosis Evaluation of plasma-membrane permeability and DNA fragmentation Evaluation of cell s metabolic activity Nuclear Staining: Propidium Iodide, Tunel Annexin V Fluorescent evaluation of phosphatidylserine Protease activity assay (caspase 1, 3, 8 Free radical detection assay (i.e. NO) Substrate for peptidase/caspase 4

5 B.2. Proteins and Genes B.2.1. Protein Purification and Analysis Protein purification Sample preparation and Gel electrophoresis Immuno assay Protein isolation and quantification Subcellular organelles (exosomes,vesicles) preparation Immuno precipitation Co-immunoprecipitation SDS Page 2D gel electrophoresis Protein gel staining Multi Cytokine release analysis (i.e. ELISA) Morphological analysis by immunocytochemical staining B.2.2. Molecular Biology DNA purification and analysis RNA purification and analysis Genotyping Gene cloning DNA purification (gel extraction sequencing) Primer customized design and PCR Nucleic acid gel electrophoresis Specific sirna cloning RNA purification RT-PCR, Real time PCR B.2.3. Cell Staining Nucleic acid staining General staining Viability and proliferation (Hoechst Tunel, PI/DAPI, YoPro) Membrane staining (i.e. DiI, FM1-43) Organell and Protein staining Customized analysis of protein expression and localization in normal or treated condition Immunocytochemistry Customized sections prepared from specific animal models subjected to specific pharmacological treatments from different tissues B. 3. Functional Assays Single cell calcium /sodium imaging Phagocytosis Membrane permeability Electrophysiology by MEA Population HT calcium imaging Migration/chemiotaxis FACS analysis Real-time videomicroscopy observation of cell behavior 5

6 C. Specialized Assays C1. Specialized Neurologic Assay and Models Neurodegeneration Neuritogenesis Neuroinflammation Epilepsy model Ischemia model Alheimer model Parkinson model Functional assays Morphological assays Morphological assays Organelle preparation Biochemical assays Functional assays Protein isolation and quantification High content MEA recording (4075x4075 electrodes Field potential stimulation Dose -response curves Cytoskeletal alterations via immunocytochemistry Alteration of synaptic plasticity Tau phosphorylation analysis Glutamatergic/GABAergic selective contribution Ionomycin/BAPTA perturbation of intracellular calcium dynamics Filopodia formation Neurite number / axonal elongation Synapse number Spine morphology Growth cone preparation Synaptosomes preparation ROS production NO release multiplex ELISAs Membrane permeability Microvesicle shedding Migration/invasion Glutamate induced exitotoxicity in vitro model Kainic acid induced exitotoxicity in vitro model Oxygen glucose deprivation in vitro model In vitro exposure to Abeta oligomers MPP+ neuronal challenge C2. Blood Brain Barrier Model The model enables to study the impact of endothelial activation on the central nervous system in a model of in vitro blood brain barrier (BBB) using primary brain endothelial cells. Endpoint analyzed: Total ROS production BH4/BH2 ratio Cell viability Inflammation panel: IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, VEGF, TNF-α, IFN-γ, EGF, MCP-1 NADPH-dependent superoxide formation enos uncoupling BBB permeability Trans-endothelial electric resistance Zonula Occludens, Tight Junctions 6

7 C3. Metabolic Model The model recreates a metabolic condition in a model of static endothelium by high glucose levels in the culture medium. Endpoints analyzed: Total ROS production BH4/BH2 ratio Cell viability Inflammation panel: IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, VEGF, TNF-α, IFN-γ, EGF, MCP-1 NADPH-dependent superoxide formation enos uncoupling 7

8 D. Customized Platforms D.1 NEUROSCRIN BDNF screening platform (partner Dr. Enrico Tongiorgi, Univ. Trieste, BRAIN Lab) BDNF Description Brain-derived neurotrophic factor (BDNF) is the most abundant and widely expressed neurotrophin in the mammalian brain and is involved in several aspects of the nervous system development as well as of the maintenance and plasticity of neural cells during the adult life. BDNF is initially produced as a precursor (probdnf) of 32 KDa, which is cleaved by three proteases into a mature form mbdnf of 14 KDa. These two forms have a different biological function: mbdnf by activating his receptor TrkB, promotes neurons survival, differentiation, and maturation, and supports the growth of axons, dendrites, spines and formation of new synapses. probdnf, by activating the receptor p75, can promote differentiation of axons vs. dendrites in the early development; probdnf can also induce long-term synaptic depression. There is another proteolytic form of BDNF of 28KDa, called truncated-bdnf, whose function is currently unknown. BDNF as Drug Target According to studies conducted over the past fifteen years, BDNF is one of the 4 major pharmaceutical targets in psychiatric diseases, including mood, eating and stress disorders, autism and schizophrenia. In addition, BDNF is considered a target in all diseases with cognitive deficits, including Alzheimer's disease and other dementias. More recently, BDNF has been shown as the major potential therapeutic agent for mental retardation and autism spectrum disorders including Huntington's syndrome, Rett and fragile-x. Lastly, excess of BDNF synthesis is considered a possible pathogenetic aspect of epilepsy. Issues with BDNF Drug Discovery BDNF is a gene with a complex regulation. probdnf is produced from 34 different human transcripts (22 in rodents), produced by alternative splicing of 11 exons. Each of these variants is transcribed independently and shows specific tissue and cellular expression patterns. Recently it has been theorized that the different mrna variants of BDNF represent a code for the regulated expression of BDNF in specific places, times and quantities in response to different stimuli (Baj and Tongiorgi, 2007; Tongiorgi, 2008). Given the strong differences in the translatability of proteins with variations in protein production up to 30 times between the different transcripts, it is necessary to discriminate the efficacy of drugs in the production of BDNF, especially at the level of translation. 8

9 Added Value of Neuroscrin Technology Neuroscrin is a patented transcript-specific assay for BDNF translation. It allows single experiment for multiple BDNF variants in high throughput, small volume set-ups. Robustly quantitative, luciferase-based assay displays a high dynamic range of responsiveness. NEUROSCRIN provides the unique opportunity to test drugs to address pathology-specific regulation of BDNF levels starting from the different BDNF isoforms. How do we meet your needs? We offer a customized screening service to identify natural or synthetic compounds that increase or decrease the translation of BDNF. In particular, we offer: A) High throughput assay to test compounds that enhance or decrease translation of BDNF from the different transcripts encoding BDNF B) Analysis of total endogenous BDNF via ELISA in primary neurons of rat hippocampus or cortex and any human cell lines with neuronal phenotype C) Analysis of total endogenous BDNF via ELISA (human, rat or mouse tissues) D) Analysis of expression of BDNF transcripts via quantitative real-time PCR (human, rat or mouse tissues or cultured cells). E) Analysis of processing of probdnf to mature and truncated BDNF by semi-quantitative Western-blotting (from human, rat or mouse tissues or cultured cells or serum). F) Neuroanatomical analysis of the expression of BDNF in the brains of animal models (semi-quantitative analysis of immunohistochemistry or in situ hybridization). G) Quantification of total BDNF serum by ELISA from patients undergoing various treatment regimens. 9

10 D.2 IVND Platform In vitro Neurodegenerative Disease Platform (partner Dr. Tiziana Borsello, Mario Negri Institute) Technology Description The IVND (In Vitro Neurodegenerative Disease) Platform represents an innovative in vitro model to study synaptopathies in different neurodegenerative scenarios. The platform is well set for Alzheimer Disease in vitro studies while is being validate for other neurodegenerative conditions (Parkinson Disease, Prions ). Presenting a number of synapses and spines tailored for each pathophysiologic condition of interest, IVND Platform enables real time analysis of a vast set of biochemical, morphological and motility information, providing a detailed plasticity profile of different region-specific primary neuronal populations. INVD Platform Key Features Working with primary living neurons, INVD Platform enables the obtaining of a relevant set of plasticity information; The evaluation of synaptic responses under different type of stimuli (stress, toxic, protection ) enables the creation of a reproducible and informative model of synaptic degeneration, dysfunction and regeneration; The real time fine controlled process allows a direct modulation of stimuli and the evaluation of resulting changes in spines parameters; The neuronal plasticity index is obtained through a fine characterization of the biochemical, morphology, motility and quantification profile of excitatory dendritic spines. Biochemical changes of spines under specific stimuli are well characterized by 3D electronic microscopy; The analysis of spine parameters is performed with a specific automated software (partner s IP) reducing operator intervention and experimental time; How do we meet your needs? IVND Platform finds potential applications in: Neuroprotection studies: screening of different compounds/drugs in a finely controlled synaptopathies scenario; Synaptic injury and dysfunction studies: synaptic degeneration profiling in specific pathologic conditions; Early diagnosis: diagnostic tool for identification of early neurodegenerative pathologic events; Morphology Studies: modulation of spine growth in neurodegenerative conditions; Biochemical Markers Analysis: studies of biochemical markers associated with synaptopathies; Chronic treatment studies; 10

11 D.3 MicroTISSUE Multiparametric Microfluidic Platform to Study the Microenvironment Technology description MicroTISSUE is a microfluidic multiparametric platform which recreates in vitro the tissue complexity by dissecting cell-cell communication and crosstalk mechanisms in specific pathophysiological scenarios. MicroTISSUE enables to culture together different cell types on separate controlled microchambers within the same chip, so that the role of each specific cell type is dissected. A specific design of the microfluidic device allows to perform tests with cells isolated from tissue in order to reach a very significant response from a biological point of view. Cells are stimulated in a highly controlled in vitro setting; multiple morphological, molecular, biochemical and functional quantitative parameters are simultaneously obtained in a time-lapse mode. By the use of physiologically relevant cell system, MicroTISSUE represents a much more detailed and physiologically relevant validation tool, which enables to better filter candidate molecules and enables early drop out of false candidates. MicroTISSUE represents an innovative platform for lead optimization services. MicroTISSUE is also very effective in the development of compounds for Rare Diseases as it is able to work with a very limited number of cells thanks to the microscale approach. The main benefit is the overall cost and time saving as well as the wealth and quality of data obtainable per experimental session, enabling drug developers and researchers to tailor much more efficiently the downstream phases of research. MicroTISSUE Key Features MicroTISSUE is designed with several microfluidic connected microchambers, where different cell populations can be cultured at the same time recreating a tissue-like cell-cell cross-talk scenario; The tool is well validated for the use of Primary Human Cells, allowing the obtaining of a pretty unique set of relevant information; Cell-cell cross-talk mechanisms after specific single/multiple/parallel stimuli can be finely analyzed in real time by: o Co-culturing different cells in a single chamber to evaluate direct physical interactions; o Culturing different cells in different chambers microfluidically connected to evaluate soluble factor-mediated interactions; The great versatility of MicroTISSUE allows its application in several ambits of the R&D process and its customization to fit specific biological needs. Working in microscale, MicroTISSUE allows the use of samples in very low concentration, reducing experimental costs and extending its application also on conditions where samples are hard to obtain; 11

12 How do we meet your needs? Main fields of application of MicroTISSUE are: 1. Research: In vitro investigation on specific cell population role and cell-cell cross-talk mechanisms in specific pathophysiological scenarios. 2. Drug Discovery & Development: Discovery: fine analysis of efficacy, toxicity, metabolism and compound-receptor binding effects and cells mechanisms after candidate drugs stimuli; Preclinical: effective selection, optimization and ranking of interesting candidates per indication; 3. Compound Repositioning: A deep understanding of the microenvironment s complexity and a specific dissection of communication mechanisms among different cell populations within a tissue of interest enables to associate existing compounds/drugs to new disease indications in a very effective way. MicroTISSUE allows an efficient and low cost repurposing of existing compounds or failed candidates, recreating in vitro the complexity mechanisms related to a physiopathologic scenario of interest and providing an informative profile of drugs/candidate activities in this context. 4. Personalized Medicine: MicroTISSUE recreates pathophysiological scenarios with a patient s cells in order to identify the most promising drugs, helping physicians to undertake a more effective patient-specific therapeutic strategy. 12

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