eoe sub articles

EoE Sub Articles

1. Fabrication of polydimethylsiloxane (PDMS) stamps
<ol>
	<li>Draw an array of microscale rings using CAD software, with an inner diameter of 250 &#181;m and an outer diameter of 450 &#181;m. The pattern used in this protocol is a 10 x 10 array with an 850 &#181;m distance between rings.</li>
	<li>Print a transparency mask of the pattern at the desired resolution using a microfabrication company&#39;s mask printing service (see Table of Materials). 
	NOTE: The provided dimensions...

ring-shaped micropatterning cell chirality assay: an in vitro technique to determine multicellular chirality based on cell alignment on ring-shaped micropatterns

Source: Zhang, H. et al. <a href="https://www.jove.com/v/63105">A Micropatterning Assay for Measuring Cell Chirality.</a> J. Vis. Exp. (2022)
In this video, we describe a cell chirality assay to determine the alignment bias of cells in a confined geometric boundary such as a ring micropattern. Chirality is an inherent property of most cell types and is determined by genetic and environmental factors. Knowing the chirality of a normal cell population can help to screen drug-treated cells.

<img alt="Figure 1" src="/files/ftp_upload/21153/21153_Figure1.jpg" /> 
Figure 1. Schematic of cellular micropatterning. (A) Procedure of microfabrication and microcontact printing for cell patterning. A negative photoresist mold was made by ultraviolet (UV) crosslinking of photoresist via a mask containing micropatterning features (1-2). Polydimethylsiloxane (PDMS) elastomeric prepolymers were cast onto the mold to create stamps (3-4). Then, an adhe...

Ring-Shaped Micropatterning Cell Chirality Assay: An In Vitro Technique to Determine Multicellular Chirality Based on Cell Alignment on Ring-Shaped Micropatterns

Source: Zhang, H. et al. A Micropatterning Assay for Measuring Cell Chirality. J. Vis. Exp. (2022)
In this video, we describe a cell chirality assay to ...

To determine in vitro mammalian cell chirality - the left-right bias of cells to align in a leftward or rightward orientation under confining conditions - begin with custom-fabricated gold-coated slides in a media-containing multi-well plate. The slides comprise micropatterns of ring-shaped islands of cell adhesion-promoting fibronectin, backfilled with cell adhesion-resistant self-assembled monolayer.
Next, seed an appropriate volume of mammalian cell suspension onto the slides and incubate. The cells adsorb onto the fibronectin-containing ring micropatterns. The surrounding self-assembled monolayer prevents non-specific cell attachment outside the ring boundaries.
Replace the media, removing any unattached cells, with fresh media. Add an actin-interfering drug to one set of slides and incubate.
The adhered cells proliferate, spreading and assuming the shape of the underlying fibronectin islands, whilst reorganizing their actin fibers in response to the spatial cues, which confine the cell monolayer, promoting biased alignment.
Now, treat the slides with paraformaldehyde for cell fixation. Under phase-contrast microscopy, the cells on the ring micropatterns appear as dark regions with bright contours aligned according to their preferential bias.&#160;
Using software, calculate each cell&#39;s angle of alignment as the deviation from the ring&#39;s circumferential direction. Average to determine the mammalian cell chirality. Actin-interfering drug-treated cells may exhibit reverse chirality.&#160;&#160;

ring-shaped-micropatterning-cell-chirality-assay-an-vitro-technique

Research

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Biochemical assays

In this video, we describe a cell chirality assay to determine the alignment bias of cells in a confined geometric boundary such as a ring micropattern. Chirality is an inherent property of most cell types and is determined by genetic and environmental factors. Knowing the chirality of a normal cell population can help to screen drug-treated cells.

Overview

Protocol

<img title="figure-representative results-58" alt="figure-representative results-58" src="/files/ftp_upload/21153/21153_Figure1.jpg" /> 
Figure 1. Schematic of cellular micropatterning. (A) Procedure of microfabrication and microcontact printing for cell patterning. A negative photoresist mold was made by ultraviolet (UV) crosslinking of photoresist via a mask containing micropatterning features (1-2). Polydimethylsiloxane (PDMS) elastomeric prepolymers were cast onto the mold to create stamps (3-4). Then, an adhe...

Ring-Shaped Micropatterning Cell Chirality Assay: An In Vitro Technique to Determine Multicellular Chirality Based on Cell Alignment on Ring-Shaped Micropatterns

In This Article

Overview

Protocol

Representative Results