Mason, D. | bioRxiv Science

bioRxiv2018

4D imaging and analysis of multicellular tumour spheroid cell migration and invasion

Studying and characterising tumour cell migration is critical for understanding disease progression and for assessing drug efficacy. Whilst tumour cell migration occurs fundamentally in 3 spatial dimensions (3D), for practical reasons, most migration studies to date have performed analysis in 2D. Here we imaged live multicellular tumour spheroids with lightsheet fluorescence microscopy to determine cellular migration and invasion in 3D over time (4D). We focused on glioblastoma, which are aggressive brain tumours, where cell invasion into the surrounding normal brain remains a major clinical challenge. We developed a workflow for analysing complex 3D cell movement, taking into account migration within the spheroid as well as invasion into the surrounding matrix. This provided metrics characterising cell motion, which we used to evaluate the efficacy of chemother-apeutics on invasion. These rich datasets open avenues for further studies on drug efficacy, microenvironment composition, as well as collective cell migration and metastatic potential.

cell biology↗

bioRxiv2016

Evaluation of quantum dot conjugated antibodies for immunofluorescent labelling of cellular targets

Semiconductor quantum dots (Qdots) have been utilised as probes in fluorescent microscopy and provide an alternative to fluorescent dyes and fluorescent proteins, due to their brightness, photostability, and the possibility to excite different Qdots with a single wavelength. In spite of these attractive properties, their take up by biologists has been somewhat limited and only a few Qdot conjugates are commercially available for the labelling of cellular targets. Although, many protocols have been reported for the specific labelling of proteins with Qdots, the majority of these relied on Qdot-conjugated antibodies synthesised specifically by the authors and therefore not broadly available, which limits the scope of applications and complicates replication. Here, the specificity of a commercially available Qdot conjugated secondary antibody (Qdot-Ab), for different antigens, was tested. Antigens were labelled simultaneously with a fluorescent dye coupled to a secondary antibody (Dye-Ab) and the Qdot-Ab. Although, the Dye-Ab labelled all of the intended target proteins, the Qdot-Ab only bound to some of the protein targets in the cytosol and could not reach the nucleus even after extensive cell permeabilisation.

biochemistry↗