Geisterfer ZM et al. (2020), Microfluidic encapsulation of Xenopus laevis ce...

XB-ART-57666

STAR Protoc 2020 Oct 31;13:100221. doi: 10.1016/j.xpro.2020.100221.

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Microfluidic encapsulation of Xenopus laevis cell-free extracts using hydrogel photolithography.

Geisterfer ZM , Oakey J .

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Cell-free extract derived from the eggs of the African clawed frog Xenopus laevis is a well-established model system that has been used historically in bulk aliquots. Here, we describe a microfluidic approach for isolating discrete, biologically relevant volumes of cell-free extract, with more expansive and precise control of extract shape compared with extract-oil emulsions. This approach is useful for investigating the mechanics of intracellular processes affected by cell geometry or cytoplasmic volume, including organelle scaling and positioning mechanisms. For complete details on the use and execution of this protocol, please refer to Geisterfer et al. (2020).

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Species referenced: Xenopus laevis
Genes referenced: dmd.2 dmd.3

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	FigureÂ 1. Preparation of microfluidic devicesSchematic showing (A) PDMS poured onto a silicon wafer containing multiple replicates of a microfluidic chamber positive and (B) device assembly after the PDMS has cured and a single chamber replicate has been cut from the curedÂ slab.
	FigureÂ 2. Successive steps for the microfluidic encapsulation of cell-free extracts using hydrogels(A) Photo-patterning of the coverslip surface in the microfluidic chamber using the DMD. A transmitted-light image of a hydrogel micro-enclosure is shown at the bottom in red boxes.(B) Cell-free extract is pumped into the device using the inlet nearest the micro-enclosures. The flow through (large collection of extract on the left side of the device) is wiped away. Red ROI indicates the position of the filled micro-enclosure which is visualized below by a fluorescent image showing the cell-free extract (supplemented with a soluble fluorophore) contained within and surrounding the hydrogel micro-enclosure.(C) After filling the micro-enclosures, an oil crossflow from the opposite inlet is used to isolate the micro-enclosure. After encapsulation, the fluorescent signal surrounding the hydrogel micro-enclosure is lost (bottom-right image). Scale bar, 50Â Î¼m. (Geisterfer etÂ al., 2020).
	FigureÂ 3. Hydrogel feature resolution(A) Digital mask drawn using Canvas 11 and imported into the Mightex software to control the DMD.(B) Transmitted-light image showing a hydrogel structure formed while the focal plane was just above the coverslip. The bottom schematic shows the position of the focal plane (in red) relative to the glass coverslip (dark blue).(C) Hydrogel structure formed with the focal plane above the cover slip surface (microfluidic chamber bottom). This result is similar for focal planes used below the coverslip surface. Both images were collected at the same axial position for direct comparison. Scale bar, 50Â Î¼m.

References [+] :

Debroy, Structured Hydrogel Particles With Nanofabricated Interfaces via Controlled Oxygen Inhibition. 2019, Pubmed