BIOTECHNOLOGY 63
In this manner, each captured
image of wells containing cells and ECM treated with various concentrations of the inhibitor compound KU-0063794, or untreated wells, over the 48-hour imaging period, was subject to preprocessing prior to placing object masks over the area (images not shown). Over time, cells and ECM in untreated wells are seen to migrate away from the original printed area to cover an increasing portion of the microplate well. Tis same migration is completely inhibited by the highest tested concentrations of KU-0063794, confirming the compound’s ability to inhibit cell signalling pathways leading to mTOR activation. Additionally, the area covered
by each 3D combination of cells and ECM was automatically calculated by Gen5 software, and returned as a calculated metric at each timepoint for the various compound concentrations tested. Tis data, along with fold change values compared to original coverage area at time 0, were then graphed as seen in Fig. 2. Te data aligns with the phenotypic image findings, and confirms the dose-dependent effect of KU- 0063794 on the migratory ability of HCT116 cells.
Summary Incorporating an automated 3D cell culture workflow in colorectal cancer research allows
Fig. 2. Kinetic HCT116 cell migration analysis. Coverage area of cells exposed to 0-10,000 nM (A) KU-0063794; and (B) KU- 0063794 area fold change calculations compared to coverage area at time 0
increased throughput and reduced human subjectivity in an environment that more closely resembles that found in vivo. A magnetic 3D bioprinting process, such as the BiO Assay Kit, provides a biomimetic 3D structure with essential cell-cell and cell-ECM communication pathways, while a multi-
mode microplate reader with automated digital imaging and advanced analysis capabilities, such as the Cytation 5, simplifies the experimental process by generating and analysing reproducible data in a single instrument.
For more information ✔ at
www.scientistlive.com/eurolab
Brad Larson is with BioTek Instruments and Glauco Souza is with Nano3D Biosciences. For more information visit
www.biotek.com
New kits for release of O-linked glycans L
udger now offers two Ludger Liberate kits that can be used to release of O-glycans from glycoprotein therapeutics; the Hydrazinolysis kit, LL-Hydraz-A2 and the Orela kit, LL-Orela-A2. Whilst hydrazinolysis is the gold standard method to remove all O-links, the Orela Kit contains reagents that are safer and much easier to handle.
O-glycans released using either of
these kits have free reducing termini so are compatible with reducing-end labelling using reagents such as 2-aminobenzamide, 2-aminobenzoic acid and Procainamide, allowing UHPLC with fluorescent detection. The Orela kit can be used for up to
12 samples. Each kit includes LC-CEX cation exchange cartridges for O-glycan purification prior to fluorescent labelling.
The hydrazinolysis kit can be used for up to 12 samples. The release conditions can be optimised for release of N-glycans, O-glycans or both. Each kit includes LC-CEX cation exchange cartridges for O-glycan purification and also LC-EB20 cartridges for N-glycan purification.
For more information visit
www.ludger.com
www.scientistlive.com
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