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:

(DS3*(((DS1-DS2)/2)+(DS2/2)))/100
 
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: http://photojournal.jpl.nasa.gov/archive/PIA20009_1997vs2015-animated.gif.

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

Wednesday, November 18, 2015

My Love Affair with the Spa; or How I learned to get my Life Back

For a number of years, I have been interested in the mammalian cell cycle. As a graduate student I worked on the relationship between the enzyme HMG CoA reductase and DNA synthesis.  HMG CoA reductase is the rate limiting step for cholesterol biosynthesis. It’s also the enzyme target for statins, which are one of the most prescribed classes of drugs in the US. Later as a post-doc my research focused on the initiation of DNA synthesis.  More recently I have been interested in the effect anti-cancer agents have on cell cycle progression.

What that really means is that I have suffered from sleep deprivation. If you have never worked on the mammalian cell cycle you might not understand the connection between cell cycle research and sleep deprivation. You see while some cells lines have longer cycles and others shorter, the average mammalian cell cycle time is approximately 24 hours long.  If you were interested in some sort of transient cell cycle regulator your experiments often required manual intervention (i.e. reagent addition, aliquot removal, measurement taken etc.) periodically throughout the entire cell cycle. The end result is that for the duration of the experiment, which might be two complete cell cycles, sleep was a commodity in short supply. On a Monday morning my first thought was that I could go to sleep on Thursday!

Sure there was automation available to those that could afford it. Large very expensive robotic systems were the rage for a period of time. Costing hundreds of thousands of dollars, these were just a dream to most researchers. Despite their price tag, most were not a viable option for live cell assays as they were not contained in HEPA filtered sterile environments.        

By now I’m sure you are wondering how this tale of woe is going to end; fortunately there is a happy ending. Much to my relief, BioTek developed the BioSpa 8. A fully functional, environmentally controlled robot that interfaces liquid handing devices with detection devices. Much like Cinderella, this solution fits me just like her glass slipper.  The BioSpa 8 holds up to 8 microplates (6- to 384-well), controls temperature, CO2, O2 and can be also provide a humidified environment in the same way as a conventional tissue culture incubator.  A robotic gripper arm can move plates from the hotel to either a BioTek liquid hander (e.g. MultiFlo FX dispenser, EL406 washer/dispenser, or a 405TS washer) or a BioTek reader (e.g. Cytation 3/5, Synergy Neo2 or an Epoch 2).  By interfacing these two device types with a plate hotel and scheduling software, a multitude of assays can be automated without breaking the bank.  The size of the system is such that it can fit in a 6 foot biosafety cabinet, allowing sterility to be maintained for live cell based assays.

Instead of spending my days and nights in the lab running experiments I can go home knowing that the reagent will get added, the samples will be washed and the plate will read.  Yes I got my life back…

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


Thursday, October 8, 2015

Analyzing the progression of the “Blood Moon” Eclipse with Gen5 software


When given the option of witnessing a once in a life time spectacle or going to bed like a responsible adult... well... usually my inner child kicks in to make the decision, which means my "responsible adult" self gets the night off.  And so it was with the recent Blood Moon Eclipse.  How could I pass up the opportunity, even though I’d be up to the wee hours of the night??  As hard as I tried, I just couldn't repress my giddy, childlike excitement.

As a photographer and astronomy enthusiast, it was an easy decision that viewing this phenomenon by eyes alone would not be enough.  I grabbed my Meade telescope, aligned it to North Star and immediately started tracking and viewing the Supermoon, which entirely filled my field of view.  As awesome as the sight was, that was just the prelude to the main event. 

Soon the eclipse began, and inch by inch, the ominous shadow of the earth advanced across the moon.  As the onslaught continued it left a wake of darkness and, as the night progressed, a secondary wake of blood red color, which is why this phenomenon is given the name “Blood Moon”.  I alternated between viewing the event through the telescope eyepiece and also doing some prime-focus astrophotography where my camera was directly mounted to the telescope, using it as a 2000mm prime lens.  The images I saw and captured were incredible and definitely worth the next day’s yawn-inducing effect.  Below is a series of photos across two and a half hours of the event. 


As I admired this lunar show, a thought hit me: wouldn’t it be cool to measure the phase of eclipse the moon was at?  A quick mental calculation told me this would be an easy task for BioTek Cytation Gen5 Image Analysis software, using Image Statistics with a Plug and a threshold.  But that could wait until morning, *yawn*.

 
Monday morning bright and early (brighter and earlier than I wished... due to my irresponsible late night gallivanting), I converted my photos into TIF files in ImageJ and opened them in Gen5 software.  First I measured the diameter of the moon using the software's Line Profile Tool, and defined an Image Statistics Plug that would perfectly outline and measure the moon. This would measure the precise size of the non-eclipsed moon which would be my reference.  Then I imported one of the photos with the eclipse. Using the Line Profile Tool again, I determined an upper threshold intensity value that would exclude the portion of the moon with high intensity that was not yet eclipsed (a value of 50,000 intensity was used). With a quick run of the analysis, I had my answer by using the Confluence measurement in Image Statistics.  90% of the moon was eclipsed in the photo I was analyzing. In the below screenshot, the blue in the image is what is excluded from the analysis, red is what is included as the portion of the moon that is eclipsed.  I quickly imported the other images and to my delight was able to measure the eclipse progression with ease! 

Now with all the easy image analysis figured out, we have 18 years until the next Blood Moon - plenty of time to develop a Cytation telescope module for an all-in-one astrophotography solution!   ... And plenty of time for me to recover from my lost sleep...


By: BioTek Instruments, Caleb Foster, Product Manager, Development

Thursday, October 1, 2015

Cytation Cell Imaging Multi-Mode Reader: Enabling Analysis of Phenotypic and Target-Based 3D Cellular Assays for Oncology Research

Cancer, the uncontrolled growth of abnormal cells in the body, is a general term used to account for more than 100 types of disease. According to Cancer Facts and Figures 2015, published by the American Cancer Society, nearly 600,000 U.S. residents will be lost to cancer by the end of this year. Global spending on cancer medications to combat each particular disease continues to increase, and for the first time crossed the $100 billion level in 2014. While concerted efforts to discover new anticancer drugs continue, this has not translated into high levels of success for potential new drugs, with up to 95% of candidate molecules failing clinical trials due to lack of efficacy or unforeseen safety concerns.

These shortcomings are in part the result of in vitro cell-based assay models that do not represent in vivo conditions. As an example, cells grown on two-dimensional (2D) hard plastic or glass substrates are easily prepared, but may not be representative of the true in vivo cell environment. Three-dimensional (3D) cell culture methods, in comparison, provide a matrix that encourages cells to organize into structures more indicative of the in vivo environment, thereby developing normal cell-cell and cell-ECM interactions in an in vitro environment.

Even as 3D cell culture methods bring about the hope of lowering lead molecule attrition rates, they also bring new challenges, particularly for assay readout systems such as conventional PMT-based detection and cellular microscopy. For example, cells aggregated into spheroid structures, which are much smaller than the area of the well of a 96-well microtiter plate are particularly challenging to monitor with conventional PMT-based detection used in microplate readers. However, in other 3D systems that encompass the whole microplate well such as scaffold-based 3D cell culture methods, PMT-based detection, which is designed to collect as much light as possible from a microplate well may provide better assay performance.

We invite you to read the Omics Tutorial, "Analysis of 3D Cell Culture Models: Enabling Phenotypic and Target-Based Assay of 3D Cellular Structures", in the September issue of Genetic Engineering & Biotechnology News to learn more how the combined automated digital widefield microscopy and conventional multi-mode microplate reading capabilities of BioTek's Cytation 3 and Cytation 5 offer a unique flexibility to enable a wide range of 3D cell culture studies.