An Open Source Based High Content Screening Method for Cell Biology Laboratories Investigating Cell Spreading and Adhesion

Abstract

BackgroundAdhesion dependent mechanisms are increasingly recognized to be important for a wide range of biological processes, diseases and therapeutics. This has led to a rising demand of pharmaceutical modulators. However, most currently available adhesion assays are time consuming and/or lack sensitivity and reproducibility or depend on specialized and expensive equipment often only available at screening facilities. Thus, rapid and economical high-content screening approaches are urgently needed.ResultsWe established a fully open source high-content screening method for identifying modulators of adhesion. We successfully used this method to detect small molecules that are able to influence cell adhesion and cell spreading of Swiss-3T3 fibroblasts in general and/or specifically counteract Nogo-A-Δ20-induced inhibition of adhesion and cell spreading. The tricyclic anti-depressant clomipramine hydrochloride was shown to not only inhibit Nogo-A-Δ20-induced cell spreading inhibition in 3T3 fibroblasts but also to promote growth and counteract neurite outgrowth inhibition in highly purified primary neurons isolated from rat cerebellum.ConclusionsWe have developed and validated a high content screening approach that can be used in any ordinarily equipped cell biology laboratory employing exclusively freely available open-source software in order to find novel modulators of adhesion and cell spreading. The versatility and adjustability of the whole screening method will enable not only centers specialized in high-throughput screens but most importantly also labs not routinely employing screens in their daily work routine to investigate the effects of a wide range of different compounds or siRNAs on adhesion and adhesion-modulating molecules.

The Neurite Outgrowth Inhibitory Nogo-A-Δ20 Region Is an Intrinsically Disordered Segment Harbouring Three Stretches with Helical Propensity

“…Fibroblast spreading was markedly inhibited on isotopically labelled Nogo-A-Δ20 substrate, confirming intact inhibitory activity of the protein. Importantly, the IC 50 value was ~40 pmol/cm 2 , which is a typical potency for Nogo-A-Δ20-induced inhibition of 3T3 fibroblast spreading [ 37 ].…”

Nogo-A: Multiple Roles in CNS Development, Maintenance, and Disease

“…This is probably attributable to a number of variables, ranging from intrinsic and extrinsic factors such as cell type and local environment up to the mode of migration. Recent advances in systems biology (Schmandke and others 2013b) will be helpful in dissecting underlying mechanisms for Nogo-A’s modulation of adhesion and cell migration.…”

Jenkins-CI, an Open-Source Continuous Integration System, as a Scientific Data and Image-Processing Platform

“…In addition, and especially in the pharmaceutical industry, the volume of images generated has dramatically increased by the adoption of high-density plate formats and/or the emergence of instruments that generate time series and Z-stacks of image fields. 5 , 6 In such cases, the time required to process tens of thousands of images on a standalone PC workstation is prohibitive. High-performance compute (HPC) clusters can provide the required speed to satisfy these increased demands.…”