Friday, March 18, 2016

Imaging and Analysis of Genotoxicity using Single Cell Gel/Comet Assay

On March 9, I had the privilege of co-presenting a webinar with Dr. Sachin Katyal from the University of Manitoba. The focus of the webinar was genotoxicity. Genotoxicity, or DNA damage, can be caused by environmental factors, behavior choices, or chemicals. Therefore it is important to test new chemicals in a wide range of applications (drugs, pesticides, dermatological agents, food additives, etc.) to ensure they do not cause damage to DNA. Or in the case of cancer look for treatments that can repair damaged DNA repair mechanisms. While there are many different types of assays to accomplish this goal, one of the most popular and most specific techniques is the comet assay. The term comes from the visual readout of the assay that resembles a telescopic image of a tradional astronomical comet that incorporates both the comet head and a long tail of streaming particles.  Conversely, the comet assay readout is produced after cell treatment with potential DNA damaging agents, mixed with agarose and added to an appropriate slide, lysed and alkaline treated, followed by electrophoresis. Upon staining with a DNA intercalating dye, cells with high levels of DNA damage will exhibit a similar comet shape (Figure 1, close up of a comet; and Figure 2, many comets in a single experiment to provide statistical significance).  These comet images are produced by the fact that fragmented DNA runs through a gel much easier than intact DNA strands. By incorporating the Cytation Cell Imaging Multi-mode readers and Gen5 software, a process that is typically very laborious and subjective can be automated to reduce manual interventions, and also have the subjectivity removed by performing analysis of all samples using mathematical algorithms. The final analysis method was extensively validated by comparisons to results generated using existing comet analysis software packages (Figure 3).

We were pleased to see 300 people register for the webinar, with many watching the actual event and participating in a lively question and answer session.

We invite you to download the webinar recording and published application note from BioTek’s website to learn more about our combined solution to perform these critical genotoxicity assays.

By: BioTek Instruments, Brad Larson, Principal Scientist

Tuesday, March 8, 2016

Vaccines and my Daughters

Vaccines have revolutionized medicine since their inception by Edward Jenner in 1798. Diseases such as small pox or polio which used to kill millions have almost been eradicated using vaccines.   These two along with a litany of other diseases are prevented through a series of childhood vaccinations. With three kids, my wife and I (mostly my wife) made numerous trips to the pediatrician's office in order for one or more of my kids to get their "shots".

One of the more recent advancements in vaccine therapies has been the development of vaccines towards human papillomavirus (HPV).  HPV is one of the most common viruses on the planet -- most people don't even know they have it and it is often sexually transmitted. In some women, however, it can have concerning consequences as the virus causes abnormal cells in the cervix and can lead to cervical cancer if left untreated. Currently HPV is the etiologic agent in 5% of all cancers worldwide [1]. These vaccines, marketed under the names Gardasil and Cervarix, provide immunity from about 75% of HPV strains. Most importantly is that it protects against those strains that are known to cause cervical cancer. Unfortunately this vaccine does not help those already infected with HPV and that's the problem.

High Grade Squamous intraepithelial lesion Pap Test. Courtesy of Michael Bonert  [2].

The inventor of the vaccine, Ian Frazer, explains this "It's quite simple really, most viruses kill the cells they infect, which is a nasty danger signal for the body so it turns on its defenses pretty quickly to kill it and then kill the cells making more of the virus. This process saves us from flu and a whole range of different infections." Human papillomavirus doesn't kill the cells it infects -- rather it makes them grow more. There's no danger signal to body -- all the body sees is tissue repairing itself." [3].  Because of this, Professor Ian Frazer and others are working on a vaccine that uses viral proteins that are normally on the surface of the infected cell as immunogens rather than the coat proteins of the intact virus.  It turns out that a new type of vaccine seems to work best in animal models, namely DNA vaccines.

DNA vaccines are considered to be the third generation of vaccine technology, and contain DNA coding specific proteins (antigens) from a pathogen. The vaccine DNA is injected into the cells of the body, where the "inner machinery" of the host cells "reads" the DNA and uses it to synthesize the pathogen's proteins. Because these proteins are recognized as foreign, when they are processed by the host cells and displayed on their surface, the immune system is alerted, which then triggers a range of immune responses.

While both of my daughters received the HPV vaccine as children and are most likely protected, these new vaccine technologies have the potential to save millions of lives. This technology is being used primarily in animals and currently there are no DNA vaccines approved in the US, but the few experimental trials offer substantial promise. If this can be made to work it might be a viable therapy for a number of other diseases including some cancers.

  1. Parkin et. al. (2005) Global Cancer Statistics, 2002 CA Cancer J. Clin. 55:74-108.
  2. By Nephron - Own work, CC BY-SA 3.0,  (2010)
  3. Cayla Dengate, (2016)  “Gardasil Creator Is Testing A DNA Vaccine To Wipe Out Cervical Cancer-Causing HPV Virus” Huffington Post Australia,

By: BioTek Instruments, Paul Held, PhD., Laboratory Manager

Tuesday, February 9, 2016

SLAS 2016 and BioSpa 8: A Love Story

Even though "SLAS 2016 and BioSpa 8" might not be a great title for the latest romance novel, it's a true blossoming love story we have witnessed at the SLAS meeting and exhibition that BioTek attends every year.

With the explosion of live-cell biology applications, more and more long-suffering cell biologists are banging their heads on the wall with frustration. The pitfalls of working with live cells are many, and the task of performing live-cell experiments may appear daunting. Many have been seen on the verge of losing their mind after working for days or weeks on preparation work only to see it all go in a quick twist of fate: It takes amazingly little to contaminate a cell culture, or to drop samples on the floor after a bad night. The inevitable question is: There must be a better way, right? So much pain and suffering in the name of science begs for a solution. Surely there is a cure out there; someone has already figured it out. Unfortunately, the curious scientist trying to answer this question will quickly find himself navigating between Scylla and Charybdis, for the solution is often automation so monstrous it is best left in the hands of the valiant adventurer, helped with an army of highly trained lab technicians and automation professionals. What to do? On one hand Charybdis the whirlpool monster of manual cell work which will get your head spinning with its long nights and weekends, heartbreaking failures and gnawing uncertainty (what is happening to my cells when I am not around?); On the other hand, Scylla, the 6-headed monster of robotic automation, that will demand more attention than your cells themselves, and will require that you boost your software and hardware IQ by 50 points before you even dare to approach it.

Then one fresh spring morning (well, it was actually in the dead of winter, but this is a love story, right?), something happened: A solution seemingly came out of nowhere with the soothing name of “BioSpa 8”. Wait… this can’t be automation, it is way too small… but… this is clearly not manual, there is some kind of moving arm in front. This brings us back to reality, and what we heard from visitors stopping by our booth at SLAS 2016 to look at BioSpa 8 in action. It went like this:
  • "This is such a great idea!"
  • "I just saw your ad: it's even smaller than I thought."
  • "This price includes the arm too? Nice!"
  • "Does it record all parameters over time? Great! And it sends text notifications? Wow!"
  • "That's it? You just programmed it?"
With the BioSpa 8 Automated Incubator, there is a better way! You can still choose to navigate the perilous strait between Scylla and Charybdis if you enjoy the rush and the sleepless nights. Or you can try the BioSpa way. Let simple, small bench top automation soothe you. And you can go back home (at a decent hour) with the reassuring thought that your days of "are-my-cells-going-to-make-it?" anguish are finally over.

By: BioTek Instruments, Xavier Amouretti, Manager, Product Marketing

Tuesday, January 12, 2016

Beyond the Solar System: Automated Comet Analysis

In our last comet assay blog post, ‘Beyond the Solar System: Automated Comet Assay Imaging’, we discussed how the comet assay  is used to directly quantify DNA damage in mammalian cells, and showed how to use Trevigen’s CometAssay® Electrophoresis System, 3-Well FLARE™ slides and 96-Well CometSlides with the Cytation 5™ Cell Imaging Multi-Mode Reader to easily image these comets. Here we’ll discuss how to analyze those images to quantify the extent of DNA damage. This quantification can be expressed as either the % DNA in the Tail or the Comet Tail Moment. Each method quantifies DNA damage by assessing the fluorescence from comet tails attributable to DNA fragments electrophoresing farther in the gel than intact DNA.

% DNA in Tail Calculation
This method is based on determining the relative circularity of the comet head and the total comet which may contain a comet tail if there is significant DNA damage. The following equation can be used:
This analysis works well due to the fact that the total comet becomes less circular with increasing DNA damage. In the case of little DNA damage, it is apparent that Total Comet Circularity ≈ Comet Head Circularity, thus % DNA in Tail ≈ 0%.However in the case of extensive DNA damage, Comet Head Circularity >> Total Comet Circularity and % DNA in Tail will tend towards 100%.
We used Gen5 Data Analysis Software to apply this calculation to all images, and using the 96-well CometChip calculations (Table 1) as an example, we can see that % DNA in Tail values increased appropriately with increasing etoposide treatment (T1-T3) of the healthy Alkaline CometAssay Control Cells (T0). The % DNA in Tail values are comparable to those obtained via different methods like ImageJ open source software with the OpenComet plugin.
Table 1. % DNA in tail calculations for 96-well CometChip

Gen5 results compare well to those seen using Loats Analysis software using the 96-well CometSlide (Figure 1), further confirming that Cytation 5 and Gen5 deliver accurate results regardless of the configuration.
Figure 1. Percent DNA in tail calculations for 96 well CometSlide using Alkaline CometAssay Control Cells in the standard comet assay

Comet Tail Moment
Another comet evaluation calculation is the Comet Tail Moment (Figure 2), which takes into account the Total Comet Length (DS1), Comet Head (DS2) and % DNA in Tail (DS3) as represented in the following equation:

Figure 2. Comet areas included in comet tail moment calculation
Comets exhibiting little to no DNA damage will have a Comet Tail Moment value approaching zero, whereas those with higher damage amounts will have increasing values. Gen5 Data Analysis Software was again used for automatic calculations, and as seen in Table 2, the Comet Tail Moment values increased appropriately with increasing etoposide treatment (CC1-CC3) as compared to untreated cells. Furthermore, these results are equivalent to those obtained via accepted methods like Loats Analysis software.
Table 2. Comet Tail Moment calculations for 3 well CometSlides.

As Cytation 5 automates cellular analyses, DNA damage using the comet assay is rapid, repeatable and comparable to analyses obtained via different analyses methods. For more detail, see our application note, Automated Imaging and Analysis of a Novel Comet Assay to Enable High Throughput Genotoxicity Testing.
By, BioTek Instruments

Wednesday, January 6, 2016

El Niño

You’ve probably heard the saying “If you don’t like the weather, just wait five minutes.” In the past few weeks, I haven’t been happy with the weather. BioTek exhibited at the ASCB show in San Diego in the middle of December, where I was freezing at 57 °F! But in New Jersey at Christmas, I was too hot at 72 °F! From what I hear about this year’s El Niño, we’re in for more surprises.

According to NASA, an El Niño weather event occurs when warm water (35-38 °F higher than normal) accumulates in the Pacific Ocean. Normally, strong equatorial winds push the warm water westward but last year, these winds were weaker than usual, allowing the warm water to move north towards California and south to Chile. Not only does this affect the local waters and aquatic wildlife, but influences extreme weather patterns worldwide. Recently, news headlines warn of flooding, ice storms and droughts. There are also comparisons between the 1997 El Niño and the current El Niño with NASA releasing this satellite image:

Heat maps can provide spatial information. In the image above, we can see that the sea surface height is highest around the equator, indicative of a pile-up of warm surface water. The full animation is amazing and can be found here:

There’s also this heat map:

Well, that’s not from a satellite. In fact, it’s a 99x99 fluorescence area scan generated using a BioTek microplate reader with Gen5 software. Just like the satellite images of El Niño, it provides spatial information - in this case, where the highest levels of fluorescence are. For example, a heat map like this can show you where your GFP-transfected cells are growing within a microplate well. Maybe you already have an application where a heat map generated from an absorbance or fluorescence area scan will help?

Whenever I hear the quote "If you don’t like the weather, just wait a few minutes", I always imagine that it will get better. In our case, during El Niño conditions, it may actually get worse!

By: BioTek Instruments, Ellaine Abueg Ph.D., Product Manager, Specialist

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.
Figure 1. 3-well slide CometSlide 2.5x fluorescent images. Individual well stained comet images using
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).
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

Tuesday, November 24, 2015

Reactive Oxygen Species: a Mechanism of Action for Insulin Resistance and Type 2 Diabetes

About one third of the US adult population is defined as obese (BMI > 30 kg/m2). While not all obese adults develop type 2 diabetes, most (~80%) people suffering from type 2 diabetes are obese1. The links are clear: overeating and a sedentary lifestyle lead to obesity and in a significant number of these individuals, insulin resistance which is the hallmark for type 2 diabetes. The mechanisms producing insulin resistance have remained unclear, however. Researchers have proposed a number of possibilities, including elevated levels of fatty acids, inflammation, endoplasmic reticulum stress and oxidative stress through the production of reactive oxygen species (ROS).

In a recent study, six healthy middle-aged men were subjected to a whopping 6,000 calorie/day diet while being confined to a hospital bed2. Within 2 days of the start of the study, the men began showing signs of insulin resistance; after a week, the men demonstrated on average a 50% decrease in their insulin-stimulated glucose uptake - a clear sign of insulin resistance.  In that week, the men had gained an average of 3.5 kg - all of it fat. That fat was biopsied and tested for the possible mechanisms of insulin resistance outlined above. Only ROS production mirrored the dramatic increase of insulin resistance.   It was found that numerous proteins associated with ROS production were up-regulated along with oxidation and carbonylation of a wide range of proteins, notably of the protein GLUT4, which translocates from intracellular vesicles to the plasma cell membrane due to insulin signaling. This protein’s structure in the fat biopsies demonstrated multiple oxidation and carbonylation sites, particularly where the glucose binding site is thought to be. This would make the glucose transporter dysfunctional and thus lead to insulin resistance.

To learn more about ROS and its physiological consequences, please download our white paper An Introduction to Reactive Oxygen Species.

1. Eckel, RH et al. Diabetes Care June 2011 vol. 34 no. 6 1424-1430.
2. The 6,000-Calorie Diet

By: BioTek Instruments, Peter Banks Ph.D., Scientific Director