Hypoxia is a feature of neurodegenerative diseases, and can both directly and indirectly impact on neuronal function through modulation of glial function. Astrocytes play a key role in regulating homeostasis within the central nervous system, and mediate hypoxia-induced changes in response to reduced oxygen availability. The current study performed a detailed characterization of hypoxia-induced changes in the transcriptomic profile of astrocytes in vitro. Human astrocytes were cultured under normoxic (5% CO2, 95% air) or hypoxic conditions (1% O2, 5% CO2, 94% N2) for 24 h, and the gene expression profile assessed by microarray analysis. In response to hypoxia 4904 genes were significantly differentially expressed (1306 upregulated and 3598 downregulated, FC ≥ 2 and p ≤ 0.05). Analysis of the significant differentially expressed transcripts identified an increase in immune response pathways, and dysregulation of signalling pathways, including HIF-1 (p = 0.002), and metabolism, including glycolysis (p = 0.006). To assess whether the hypoxia-induced metabolic gene changes observed affected metabolism at a functional level, both the glycolytic and mitochondrial flux were measured using an XF bioanalyser. In support of the transcriptomic data, under physiological conditions hypoxia significantly reduced mitochondrial respiratory flux (p = 0.0001) but increased basal glycolytic flux (p = 0.0313). However, when metabolically stressed, hypoxia reduced mitochondrial spare respiratory capacity (p = 0.0485) and both glycolytic capacity (p = 0.0001) and glycolytic reserve (p < 0.0001). In summary, the current findings detail hypoxia-induced changes in the astrocyte transcriptome in vitro, identifying potential targets for modifying the astrocyte response to reduced oxygen availability in pathological conditions associated with ischaemia/hypoxia, including manipulation of mitochondrial function, metabolism, and the immune response.

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Expansion microscopy (ExM) is a versatile super-resolution microscopy pipeline, leveraging nanoscale biomolecular cross-linking and osmotically driven swelling of hydrogels. In its current implementation, ExM remains a laborious and skill-intensive technique, involving manual handling of the hydrogels that can compromise the integrity of the gel matrix and diminish reproducibility. The lack of protocols to constrain the gel orientation during this process lends to challenges in tracking gel isotropy during or after the swelling. We have developed a bespoke microplate system capable of carrying out the entire ExM workflow within each well. The microplates enable in situ image acquisition and eliminate the need for direct physical handling of the hydrogels. The preservation of the gel geometry and orientation by the design of the microplate wells also enables convenient tracking of gel expansion, pre- and post-ExM image acquisition, and distortion mapping of every cell or region of interest. We demonstrate the utility of this approach with both single-colour and multiplexed ExM of HeLa cells cultured within the microplate wells to reveal nuclear and sub-plasmalemmal regions as distortion-prone structures.

Amine-reactive esters of aromatic fluorescent dyes are emerging as imaging probes for nondescript staining of cellular and tissue architectures. We characterised the differential staining patterns of 14 fluorescent dye ester species with varying physical and spectral properties in the broadly studied human cell line - HeLa. When combined with expansion microscopy (ExM), these stains reveal nanoscale features such as the nuclear proteome, membrane-bound compartments and vesicles. Among N-Hydroxysuccinimide (NHS) esters, we observe differential compartment specificity and weighting of labelling density which correlates with the hydrophobicity of the dye ester. We also observe changes in both staining density and compartment specificity for a given dye ester depending on the presence of a second dye ester species and on the timepoint of application in the ExM protocol. Our findings confirm these dye esters as a useful addition to the repertoire of biomedical stains of the cellular proteome, either on their own, or as counterstains to immunofluorescence.