Specifically, the multi-well format allows many factors to become assayed in parallel, whilst lowering the amounts of mice necessary to provide tissues concurrently. Whilst this style of CNS light matter isn’t designed to replace all pet work, it could inform, or augment such tests. useful properties and experimental strategies, https://doi.org/10.5256/f1000research.16802.d233269 ( Bijland transgenic myelin or latex beads. Body 4C C scans of X-ray movies of proteome arrays pursuing treatment for seven days with PBS, latex beads, outrageous type myelin or tg myelin, plus raw values in arbitrary units and normalised values (to positive control spots) for spot intensity for each of the cytokines assayed, plus a template for cytokine array. Physique 5 – scans of X-ray films and raw arbitrary unit values of band sizes and intensities. Physique 6 and 7 C images from IN Cell Analyzer 2000 and CellProfiler values for myelin, axons and DAPI +ve nuclei. Due the size of files for the microplate images these can not be provided Febuxostat (TEI-6720) but are available at request from the corresponding author (JE) ku.ca.wogsalg@ragde.ailuj Peer Review Summary models and clinical trials. However, models that replicate the cellular complexity of the CNS can inform these approaches, reducing costs and minimising the use of human material or experimental animals; in line with the principles of the 3Rs. Using electrophysiology, pharmacology, time-lapse imaging, and immunological assays, we demonstrate that murine spinal cord-derived myelinating Febuxostat (TEI-6720) cell cultures recapitulate spinal-like electrical activity and innate CNS immune functions, including responses to disease-relevant myelin debris and pathogen associated molecular patterns (PAMPs). ?Further, we show they are (i) amenable to siRNA making them suitable for testing gene-silencing strategies; (ii) can be established on microelectrode arrays (MEAs) for electrophysiological Febuxostat (TEI-6720) studies; and (iii) are compatible with multi-well microplate formats for semi-high throughput screens, maximising information output whilst further reducing animal use. We provide protocols for each of these. Together, these advances increase the utility of this tool for studying normal and pathological development and function of white matter, and for screening therapeutic molecules or gene targets for diseases such as multiple sclerosis, motor neuron disease or spinal cord injury, whilst avoiding approaches on experimental animals. situation. Uncouples CNS-intrinsic responses from those mediated by peripheral organs and systems. Ease of manipulation, i.e. genetic using siRNA; neuronal electrical activity using pharmacological modulators of neural activity; small molecules using small molecule libraries; relevant PAMPs or DAMPS. Can be generated from transgenic reporter mice for live imaging and functional readouts or from mutant or transgenic models of disease. 3Rs benefits:?Inform studies and/or acts as an adjunct to studies using models of multiple sclerosis, motor neurone disease, the leukodystrophies and other neurodegenerative diseases involving white matter, minimising the use of experimental animals and maximising information obtained. Practical benefits:?Straightforward to establish in any lab with cell culture facilities. Compared to CNS slice cultures, which also contain all major neural cell types, this cell culture system is easier to maintain (oxygen and nutrients readily reach all cells) and quicker to set up. Cells can be grown on microelectrode arrays that do not require the specialist expertise needed for single cell electrophysiology. Cells can be grown on multi-well plates for semi-high throughput assays. Multi-well microplate formats facilitate the testing of multiple factors on parallel cultures. Current applications:?Semi-high throughput screens for pro-myelinating or inhibitory factors; testing and verifying siRNA constructs and/or testing the consequences of gene knockdown; modulating neural activity to assess secondary consequences for other cells types; live imaging of cellular interactions. Potential applications:?Semi-high throughput screens for factors that affect axonal survival or regeneration. Assessing effects of microglial ablation on other cell types, by manipulating CSF1R signalling pathway. Live imaging of organelle distribution and/or transport. Initial screen of anti-sense oligonucleotides for gene silencing in the context of techniques, such as human-derived induced pluripotent stem cell models or murine cell cultures. Indeed, cell and tissue culture is used widely in neuroscience to study the development and function of the major cell types of the CNS; Rog neurons, oligodendroglia, astrocytes and microglia. The main reasons being, models are a) relatively inexpensive; b) amenable to manipulation, including pharmacological and genetic; c) accessible to live imaging and optogenetic approaches; Febuxostat (TEI-6720) d) reduce the reliance on experimental animal models for early-stage screening/proof-of-concept studies; and e) inform Febuxostat (TEI-6720) subsequent animal studies, if they are required. In particular, cell culture assays can reduce the numbers of animal used in studies by, for example (i) guiding selection of candidate therapeutic molecules, (ii) informing drug dosage concentrations, (iii).