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The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.
Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular compartments while circumventing complications arising from natural cell-to-cell variations. To standardize cell shape, cells are either confined in 3D molds or controlled for adhesive geometry through adhesive islands. However, traditional micropatterning techniques based on photolithography and deep UV etching heavily depend on clean rooms or specialized equipment. Here we present an infrared laser assisted micropatterning technique (microphotopatterning) modified from Doyle et al. that can be conveniently set up with commercially available imaging systems. In this protocol, we use a Nikon A1R MP+ imaging system to generate micropatterns with micron precision through an infrared (IR) laser that ablates preset regions on poly-vinyl alcohol coated coverslips. We employ a custom script to enable automated pattern fabrication with high efficiency and accuracy in systems not equipped with a hardware autofocus. We show that this IR laser assisted micropatterning (microphotopatterning) protocol results in defined patterns to which cells attach exclusively and take on the desired shape. Furthermore, data from a large number of cells can be averaged due to the standardization of cell shape. Patterns generated with this protocol, combined with high resolution imaging and quantitative analysis, can be used for relatively high throughput screens to identify molecular players mediating the link between form and function.
Cell shape is a key determinant of fundamental biological processes such as tissue morphogenesis1, cell migration2, cell proliferation3, and gene expression4. Changes in cell shape are driven by an intricate balance between dynamic rearrangements of the cytoskeleton that deforms the plasma membrane and extrinsic factors such as external forces exerted on the cell and the geometry of cell-cell and cell-matrix adhesions5. Migrating mesenchymal cells, for instance, polymerize a dense actin network at the leading edge that pushes the plasma membrane forw....
1. Coverslip preprocessing
The quality of the experimental data obtained through micropatterning is largely dependent on the quality of the patterns. To determine the quality of patterns generated with the method above, we first used reflectance microscopy to assess the shape and size of the photo ablated areas of the coverslip. We found that each individual pattern looked very similar to the ablation mask and displayed clear boarders and a surface that reflected light uniformly (Figure 2B). A variety of shapes a.......
The results above demonstrate that the described IR laser assisted micropatterning (microphotopatterning) protocol provides reproducible adherent patterns of various shapes that enables the manipulation of cell shape and cytoskeletal architecture. Although numerous micropatterning methods have been developed both prior to and after the debut of microphotopatterning, this method possesses several advantages. First, it does not require specialized equipment and cleanrooms that are usually only found within Engineering depa.......
This work was supported by Connaught Fund New Investigator Award to S.P., Canada Foundation for Innovation, NSERC Discovery Grant Program (grants RGPIN-2015-05114 and RGPIN-2020-05881), University of Manchester and University of Toronto Joint Research Fund, and University of Toronto XSeed Program. C.T. was supported by NSERC USRA fellowship.
....Name | Company | Catalog Number | Comments |
(3-Aminopropyl)trimethoxysilane | Aldrich | 281778 | |
10 cm Cell Culture Dish | VWR | 10062-880 | Polysterene, TC treated, vented |
25X Apo LWD Water Dipping Objective | Nikon | MRD77225 | |
3.5 cm Cell Culture Dish | VWR | 10861-586 | Polysterene, TC treated, vented |
4',6-Diamidino-2-Phenylindole (DAPI) | Thermo | 62248 | 1mg/mL dihydrochloride solution |
Bovine Serine Albumin | BioShop | ALB005 | |
Dulbecco's Phosphate-Buffered Saline | Wisent | 311-425-CL | |
Ethanolamine | Sigma-Aldrich | E9508 | |
Fibronectin | Sigma-Aldrich | FC010 | 1mg/mL in pH 7.5 buffer |
Fibronectin Antibody | BD | 610077 | Mouse |
Fiji | ImageJ | Version 1.53c | |
Fluorescent Phalloidin | Invitrogen | A12380 | 568nm |
Glass Coverslip | VWR | 16004-302 | 22 × 22 mm |
Glutaraldehyde | Electron Microscopy Sciences | 16220 | 25% aqueous solution |
Hydrochloric Acid | Caledon | 6025-1-29 | 37% aqueous solution |
IR Laser | Coherent | Chameleon Vision | |
Minimal Essential Medium α | Gibco | 12561-056 | |
Mounting Medium | Sigma | F4680 | |
Mouse Secondary Antibody | Cell Signaling Technology | 4408S | Goat, 488nm |
Multi-Photon Microscope | Nikon | A1R MP+ | |
Myosin Light Chain Antibody | Cell Signaling Technology | 3672S | Rabbit |
NIS Elements | Nikon | Version 5.21.03 | |
Nitric Acid | Caledon | 7525-1-29 | 70% aqueous solution |
Photoshop | Adobe | Version 21.2.1 | |
Pluronic F-127 | Sigma | P2443 | Powder |
Poly(vinyl alchohol) | Aldrich | 341584 | MW 89000-98000, 98% hydrolyzed |
Rabbit Secondary Antibody | Cell Signaling Technology | 4412S | Goat, 488nm |
Shaker | VWR | 10127-876 | Alsoknown as analog rocker |
Sodium Borohydride | Aldrich | 452882 | Powder |
Sodium Hydroxide | Sigma-Aldrich | S8045 | |
Sodium Phosphate Dibasic | Sigma | S5136 | Powder |
Sodium Phosphate Monobasic | Sigma | S5011 | Powder |
Spyder | Anaconda | 4.1.4 | |
Trypsin | Wisent | 325-042-CL | 0.05% aqueous solution with 0.53mM EDTA |
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