Wednesday, December 16, 2015

Beyond the Solar System: Automated Comet Imaging

Witnessing a comet streak through space is spectacular; and for ages, they’ve been analyzed to help unlock some of the universe’s great mysteries. Here on terra firma, we can use a much smaller type of comet to unlock some of the great mysteries of cellular metabolism.

The comet assay is used to directly quantify DNA damage in mammalian cells. Knowing the extent of damage that a compound can have on DNA has huge implications in epidemiology, toxicology, drug development and more. The principle is straightforward, where cells are embedded in agarose gel, treated, lysed, electrophoresed, stained and fluorescently imaged. Undamaged DNA moves very slowly through the gel, but when fragmented, the damaged DNA is able to move through the gel pores at a faster rate. When fluorescently imaged, they resemble green comets – significantly smaller than their interstellar namesakes, but equally as interesting, we think!

Manual methods of this sensitive assay limit throughput, but thanks to our Cytation 5™ Cell Imaging Multi-Mode Reader and our friends at Trevigen, we validated ways of automating assay imaging and analysis so that you can achieve increased sample throughput with decreased manual variability. Here, we’ll focus on imaging, and in a subsequent blog, we’ll discuss how to analyze these comet images.

Automated Slide Imaging

We used Trevigen's CometAssay® Electrophoresis System, 3-Well FLARE™ slides, 96-Well CometSlides and other components to run an alkaline comet assay using Trevigen’s Alkaline CometAssay Control Cells that were treated with and without etoposide, a compound known to damage DNA. Prior to imaging with Cytation 5’s GFP imaging channel, we placed the 3-well slides into a slide adapter. For higher throughputs, we used the 96-well CometSlide, and a different, two-part adapter, to ensure consistent positioning. For higher throughputs with varying treatments or cell types, scroll down to Automated Chip Imaging where we discuss Trevigen's CometChip® System. All slide wells were automatically imaged. Since 3-Well CometSlides cover such a large area, Cytation 5 captured individual 2.5x images and stitched them together in a 4x3 image montage, providing an extended field of view of 8165 x 7956 µm. Conversely, the area of the agarose gel contained in individual wells of the 96-Well CometSlides was small enough such that the field of view provided by the 2.5x objective was sufficient to capture 100 - 120 comets/well.

If we auto-focus on 3-Well CometSlide individual images from the montage (Figure 1), the extent of DNA damage from etoposide treatment becomes clear. The untreated comets are well-defined spheres/dots/asteroids, while the etoposide-treated comets show a distinct comet tail where the DNA fragments moved faster than the intact DNA.

3-well slide CometSlide 2.5x fluorescent images
Figure 1. 3-well slide CometSlide 2.5x fluorescent images. Individual well stained comet images using cryopreserved Alkaline CometAssay Control Cell populations, revealing high (+etoposide) or no appreciable DNA damage above normal levels (-etoposide).

Automated Chip Imaging

The 96 Well CometChip System from Trevigen offers benefits beyond the high-throughput format of their 96 well CometSlide. The system allows simultaneous treatment and measurement of DNA damage by varying treatments, or among different cell types, on a single slide. Using this system, cells are captured by gravity into micropores created in a thin layer of agarose, forming an organized grid pattern. This evenly distributes comets throughout the well, increasing simplicity and accuracy (Figure 2).

96-well CometChip 2.5x fluorescent images
Figure 2. 96-well CometChip 2.5x fluorescent images. Images captured of wells demonstrating high (+etoposide) or no appreciable DNA damage above normal levels (-etoposide)
 
When comparing Figure 2 to Figure 1, one can clearly see that the random comet placement is eliminated using the CometChip system, thus simplifying automated analysis and reducing the number of objects requiring removal before final calculations were performed. Additionally, if you compare the comet configurations closely, you’ll see that Figure 2 comet tails appear on the left, while Figure 1 comet tails appear on the right. This is because we opted to place the CometSlides, with dried agarose, in a typical slide configuration where the stained wells faced down, and the CometChip, with wet agarose, in an upward facing orientation. These mirrored configurations do not affect analyses.

Cytation 5 automates sample translation, focusing and image acquisition enabling the detection of small increases in comet tail fluorescence. Additionally, single or image montage capture allows automated procedures to be performed with all CometAssay configurations.

So now that we automated comet image acquisition, how is this used to calculate DNA damage? Stay tuned for the next installment to see how Cytation 5 easily automates comet assay analysis. And if you want all the details, see our application note, Automated Imaging and Analysis of a Novel Comet Assay to Enable High Throughput Genotoxicity Testing.


By, BioTek Instruments

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