Cell migration plays a key role in development, tissue repair, immune function, angiogenesis, and metastasis. A major in vitro assay for quantifying cell migration is the scratch assay, in which a confluent layer of tissue culture cells is scratched and the extent to which the wound is filled in over time by surrounding cells through migration is measured by light microscopy. In order to make this assay more reliable and reproducible, and to allow it to be automated for high-throughput screening, T. Gebäck, M.M.P. Schulz, P. Koumoutsakos, and M. Detmar at the Swiss Federal Institute of Technology (Zurich, Switzerland) have devised a novel automated image analysis technique called TScratch for identifying and quantifying the open area of the scratch using a recently developed edge-detection algorithm. This freely available software, implemented as a MATLAB GUI and as a standalone package, allows any set of images in standard file types such as TIFF and JPEG to be automatically and rapidly analyzed, but also provides the flexibility to customize the analysis through the elimination of images with poorly delineated open areas or the manual modification of the open area in an image using various tools. The measured open areas of the all the analyzed images, along with various computations such as means and standard errors, can be obtained as an output file for importation into a spreadsheet program for further analysis. The authors examined scratch assays of various cell lines and demonstrated that the manual and TScratch results closely matched, but that the software completed the analysis five times faster with minimal user intervention.

(See “TScratch: A novel and simple software tool for automated analysis of monolayer wound healing assays” on page 265.)

Beam Waist Slimmed by TSLIM

Three-dimensional (3-D) imaging provides valuable information for understanding structure and function relationships. To obtain these images, tissues must be sectioned, either mechanically by routine histological procedures, or nondestructively via optical sectioning. Confocal and multiphoton microscopy produce high-resolution aligned sections, but only in samples of a few hundred microns or less in thickness. Thicker specimens may be imaged by magnetic resonance histology (MRH) or micro-computed tomography (CT), but at much lower resolution. Light sheet–based microscopy offers another option for nondestructive optical sectioning of thick tissues. This is accomplished by projecting a thin light sheet with a cylindrical lens through a specimen to illuminate an optical section within the tissue. A z-stack of serial sections can be produced by moving the light plane through the specimen and observing it orthogonally. Thin-sheet microscopy allows nondestructive sectioning of transparent or fixed cleared thick tissues at higher resolution than MRH or CT, but the necessary microscope is not commercially available. Researchers at the University of Minnesota, Minneapolis led by J. Leger have collaborated with M. Hillenbrand at the Technische Universitat in Ilmenau, Germany to develop a thin-sheet laser imaging microscope (TSLIM) for nondestructive optical sectioning of thick tissues. The microscope is modular, allowing for configurations with different lasers, beam expanders, lenses, and specimen chambers, and could obtain greater resolution of small structures by narrowing the beam waist. They report detailed information for assembly of the device and display its capabilities with images of mouse cochlea, zebrafish brain and inner ear, and rat brain.

(See “Thin-sheet laser imaging micro-scopy of optical sectioning of thick tissues” on page 287.)

Getting the HeLa out of There

Recently published evaluations of the costs of using unauthenticated cell lines have increased researcher attention to the issue of cell line contamination. It is estimated that 18% of cell lines submitted to repositories are contaminated with other cell types, with aggressively growing HeLa cells responsible for 25% of these contaminations. Occasionally, the contaminating cell line can take over a culture, resulting in the loss of valuable research material. The increased focus on this issue has led to calls for mandatory cell line monitoring prior to publication or grant funding and increased awareness of the need for sensitive cell identity assays to fulfill these requirements once they are established. To assist in this effort, members of R. Badge's laboratory at the University of Leicester have developed an inexpensive and rapid PCR-based HeLa-specific genotyping assay. This single duplex PCR detects a full-length L1 retrotransposon that is specific to HeLa cells. The researchers found that this particular insertion was present in HeLa cells acquired from geographically different sources in Europe and the USA and absent from human DNA samples from diverse populations. The assay is sensitive to low levels of HeLa contamination, detecting 1% HeLa DNA mixed with non-HeLa DNA. The assay requires only a single tube and is robust enough to correctly genotype unpurified DNA contained in frozen cell pellets. Importantly, this assay is accessible for standard molecular biology labs and less expensive than short tandem repeat (STR)–based DNA fingerprinting methods, thereby facilitating easy and routine cell line monitoring of HeLa contamination events.

(See “A novel L1 retrotransposon marker for HeLa cell line identification” on page 277.)