Assays and Assay Development
Our facility has high capacity with 120 plates (x2) at ambient conditions, 42 plates in an automated CO2 cell incubator, and 40 plates cooled. But we can also assay single 96 384 or 1536 plates as needed. We have experience with many assay formats. Some have high z’ factors well above 0.5 and are suitable for screens, whereas some functional assays are more suitable for characterisation of screen hits. Contact us (link below) to discuss your needs.
Examples of assays currently routine in our facility include:-
1. Protein Interaction Assays
A wide variety of protein interaction assays for either library screens in high-throughput (singly or multiplexed followed by deconvolution) or more limited sets. These assays use either recombinant purified proteins, cell-free conditions or in intact cells. An example of a small screen is shown. Some others can be found in references 1-4 below. We assist in library handling, assay set-up and data acquisition and interpretation according to your needs.
2. Cell viability
Cell viability assays typically evaluate nuclear morphology and membrane permeability by imaging and/or plate reader assays. Examples of dual PI/LDH analyses can be found in reference 1. The image shows an example from a multiplate 384 well assay for caspase activation in primary cultures.
3. Cell responsiveness – reporter dyes with on-stage pipetting
Secondary validation of specificity of protein interaction inhibitors may require negative screening for off-target effects. Below is an example in which compounds identified from a high-throughput screen and validated for target engagement are separately evaluated for possible off-target effect on neuronal calcium responsiveness, revealing one compound for elimination. Dye loading (in this case, a calcium dye) and on-stage cell stimulation to each well during imaging are fully automated.
4. Small animal models
Changes to cells in small animal models such as C.elegans as shown (see also reference 5) are also possible.
We are currently establishing high-throughput imaging assays applicable to zebrafish larvae (<5 dpf) in collaboration with the zebrafish core facility at the Turku Centre for Biotechnology. Our current long-term capacity is for 3 x 96 well plates imaged simultaneously. Time-projection data from single larvae in each of 6 wells are shown below as an example.
5. Combination approaches with optogenetics
Optogenetics greatly simplifies the high-throughput application of a range of conditions in a more flexible way that liquid handling alone can offer. Our facility is experienced with integration of complex signalling pathway regulation paradigms using cellular optogenetic approaches (currently focused on MAPKs, see reference 4). This approach can be combined with liquid handling if needed, and synchronised with automated image acquisition or other measurements. Contact us for more details.
6. Oncology models
For oncology models please follow this link or contact Malin Åkerfelt (malake[at]utu.fi)
7. References Cited
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.
5. Lehtonen S, Jaronen M, Vehviläinen P, Lakso M, Rudgalvyte M, Keksa-Goldsteine V, Wong G, Courtney MJ, Koistinaho J, Goldsteins G (2016) Inhibition of excessive oxidative protein folding is protective in MPP+ toxicity-induced PD models. Antioxidants & Redox Signaling. 25(8): 485-497. doi:10.1089/ars.2015.6402. PMID: 27139804.