Screening Phenotypic Assays

    Viability assays are important methods in neuroscience, oncology and other fields, and are among our most requested services. The effects of compound libraries, newly developed modulator compounds, drug combinations or RNAi knockdown on specific cell-based disease models can be determined. A wide range of viability assays are available. Some assays measure ATP levels (figure A, a single synergy analysis graph extracted from a multiple cell line multi-drug combination screen), others measure the reducing capacity of cells (e.g. WST-type assays) or the release of cytoplasmic markers such as LDH (B) in cell culture wells with a plate reader. Yet others evaluate cell and tissue membrane permeability (C, organohippocampal slice culture shown, see ref 2) and/or nuclear morphology with imagers and/or plate readers. Examples of dual PI/LDH analyses can be found in reference 1. Figure D below zooms in on sub-field from a single well in a multiplate 384 well assay for caspase activation in primary neuronal culture (red indicates activated caspase is present, yellow/green shows it is not).

    Contact: Michael Courtney, michael.courtney@utu.fi

    References

    1. Li LL, Ginet V, Liu X, Vergun O, Tuittila M, Mathieu M, Bonny C, Puyal J, Truttmann AC, Courtney MJ (2013) The nNOS-p38MAPK pathway is mediated by NOS1AP during neuronal death. J Neurosci. 33, 8185-8201. doi: 10.1523/JNEUROSCI.4578-12.2013 PMID: 23658158
    2. Li LL, Melero-Fernandez de Mera RM, Chen J, Ba W, Nadif Kasri N, Zhang M, Courtney MJ (2015) Unexpected Heterodivalent Recruitment of NOS1AP to nNOS Reveals Multiple Sites for Pharmacological Intervention in Neuronal Disease Models. J Neurosci. 35, 7349-7364. doi: 10.1523/JNEUROSCI.0037-15.2015 PMID: 25972165
    3. Li LL, Cisek K, Courtney MJ (2017) Efficient Binding of the NOS1AP C-Terminus to the nNOS PDZ Pocket Requires the Concerted Action of the PDZ Ligand Motif, the Internal ExF Site and Structural Integrity of an Independent Element. Front. Mol. Neurosci. 10:58. doi: 10.3389/fnmol.2017.00058. PMID: 28360833.
    4. Melero-Fernandez de Mera RM*, Li LL*, Popinigis A, Cisek K, Tuittila M, Yadav L, Serva A, Courtney MJ (2017) A simple optogenetic MAPK inhibitor design reveals resonance between transcription-regulating circuitry and temporally-encoded inputs. (*equal contribution) Nat. Commun. 8, 15017. doi: 10.1038/ncomms15017. PMID: 28497795.
    We also offer opportunities to record changes to cells with small animal models such as C.elegans as shown (see Lehtonen et al., 2016).

    In collaboration with the zebrafish core facility at the Turku Bioscience Centre, we are expanding high-throughput methods and assays for zebrafish larvae (<5 dpf) . We have automated a staining protocol using the screening unit pipetting robotics. Automated imaging can be carried out on the High-throughput microscopes. We began to set up a high-throughput imaging assay for monitoring behaviour of zebrafish larvae (<5 dpf). Our long-term capacity is being increased from 3 x 96 well plates imaged simultaneously. Time-projection data from sample 6 wells (only single larvae in each well, movement is visualised by the appearance of larvae in multiple positions) are shown below as an example. Larvae can be exposed to reagents and libraries and/or physiological stimulus such as a flash of light, and behaviour is analysed by automated image analysis.

    Contact: Michael Courtney, michael.courtney@utu.fi

    References

    Lehtonen Š, Jaronen M, Vehviläinen P, Lakso M, Rudgalvyte M, Keksa-Goldsteine V, Wong G, Courtney MJ, Koistinaho J, Goldsteins G. Inhibition of Excessive Oxidative Protein Folding Is Protective in MPP(+) Toxicity-Induced Parkinson‘s Disease Models. Antioxid Redox Signal. 2016 Sep 10;25(8):485-97. doi:10.1089/ars.2015.6402. Epub 2016 Jun 15. PubMed PMID: 27139804.
    Details coming soon