We used laser micropatterning to fabricate monolithic hydrogel nanowells in standard microwell plates for simultaneous single cell secretion and phenotype analysis.
The release of cellular DNA as neutrophil extracellular traps (NETs) plays a pivotal role in the immune response by entrapping and killing pathogens. NET release occurs at a greater frequency within neutrophil clusters and swarms, indicating a potential for collective behaviour. However, little is known about how dense clustering of cells influences the frequency of NET release. Using an image-based assay for NETosis within nanowells, we observed that the frequency of NETosis correlated with the number of cells within the nanowell. Upon co-incubation of NETosis-induced cells with naïve neutrophils, we observed that NET release was induced in naïve cells in a cell density-dependent manner, which was potentiated by NET DNA. By embedding NETosis-induced cells within plaques, we characterized the propagation of NETosis in naïve neutrophils. NET release in naïve neutrophils was enhanced over time but remained spatially restricted to the periphery of the plaque. Together, we show that NETosis is an auto-amplified process but that the spatial propagation of NET release is strictly regulated.
We applied an unmatched back projector based deconvolution algorithm to reduce image reconstruction time in light field microscopy. 3D laser written microstructures were fabricated and used to characterize the light field imaging.
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