Friday, January 4, 2019

How Cell Counts Provide More Context to Your Data

A couple of months ago I, like most of the country, was captivated by the $1.6 billion dollar lottery drawings. After plunking down my $10, I let myself dream about hitting that jackpot and all the trappings of immense wealth. My mind drifted as I could smell the ocean standing on the lanai of my beachside home on some distant Hawaiian Island. Eventually snapped back to reality (Boston traffic does that to you quickly) – I realized I fell into a common pitfall – focusing on a result without accounting for variables. In the case of the lottery, the biggest variable being the odds of winning. With a possible 302 million combinations, the odds of hitting my numbers were about 6 times higher than getting killed by a falling coconut (1 out of 50 million).

All of this discussion of odds got me thinking about data sets I see at our customers’ sites. I often glance at heat maps of raw data as they are generated from our instruments. The high contrasting colors always look promising at first, but unlike the lottery, as scientist we are trained to account for variability. One critical variable in any cell-based assay is that differing numbers of cells between individual assay points can skew data. Typically cell cultures are optimized to account for proliferation rates, rate of cell death, cell adhesion so that cell numbers are consistent before performing any assays. Even with optimized conditions, there can be small variations that can affect the final cell density. These differences can ultimately cause increased variability across replicate data and throughout the experiment. Thus getting an accurate cell count within the assay well would allow for normalization of the data and remove this source of variability.

There are many strategies for normalizing cell population but the preferred method for normalization involves counting fluorescently stained cells in each well of a microplate via microscopy. With BioTek’s imaging and microscopy products, we provide an automated solution for cell counting that historically had been a tedious manual process or required the use of expensive imaging instruments. In the following example we use the nuclear stain DAPI to identify the cell nucleus then BioTek’s Gen5™ software automatically sets a threshold to provide accurate cell counts.

Figure 1: In-Situ Nuclear Staining (A) Threshold Analysis for object selection and count
As a Field Applications Scientist, I see firsthand how this powerful tool helps advance our customers’ research. Earlier this year, we launched a new integrated solution with Agilent Technologies that combined Agilent’s cellular metabolic analysis with BioTek’s imaging technologies. Centered around Agilent’s Seahorse XFe analyzers, customers can monitor real-time oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of live cells in a multi-well plate, interrogating key cellular functions such as mitochondrial respiration and glycolysis. When coupled with BioTek Cytation™ line, we now provide a powerful and intuitive turnkey platform for normalizing metabolic data. Perhaps the most impressive part of the system is how seamless the interface and workflow is for the customer. With a unified software look and feel provided by Agilent (with Gen5 powerful tools running in the background), the image based normalization is initiated with a simple barcode scan and a few confirmation checks. All of the data is centralized into the Agilent Wave software for complete analysis and interpretation. When the Agilent system is not active, the customer can utilize the Cytation as a stand-alone imager for other quantitative microscopy applications.

For more detailed information, see the XF Data Normalization by the Agilent Seahorse XF Imaging and Normalization System Application Note.

By: BioTek Instruments, Jared Amuan, Field Application Scientist

Tuesday, November 20, 2018

2018 SfN Meeting: Remembering Automated Microscopy in your Dementia Research

Dementia is a class of mental process disorders for which there are currently little to no treatment options. This is especially true in the later stages of Alzheimer's, a significant target for new treatments. At the 2018 Society for Neuroscience Conference, a commonly used model was presented containing three pathological components of Alzheimer's research: amyloid-β plaques, tau-containing neurofibrillary tangles, and microglia activation. In each one of these three research fields, the automated microscopy and plate reading functionality that BioTek Instruments provides will continue to play a crucial role in both qualitative and quantitative analyses. The combination of fixed and live cell samples will require both versatile and sensitive instrumentation to advance research aims.

These three Alzheimer's pathological pathways have their own unique biomarkers and analytical questions. In amyloid-β and tau neurofibrillary tangles research, a common assay presented was the measurement of the accumulation and structure of these proteins. For the larger samples of neurological tissue, both immunofluorescence and immunohistochemistry techniques continue to assess the progression of the disease such as gross production of plaques and fibrillary aberrations. At the intracellular level, microscopy is used to track molecular dynamics to understand the role of both localizations and posttranslational modifications in disease progression and treatments. The establishment of the role of microglia in Alzheimer’s disease is playing a critical role in progressing disease models and innovative treatments. Not only can fluorescent microscopy be used to examine defining cell type markers and molecular changes in fixed samples but live co-cultures are also used to examine intercellular interactions. Localizing and measuring the proximity of different cell types in addition to the simultaneous imaging of neuronal phenotypes are unraveling a complex and important story that may help many in the near future.

By: BioTek Instruments, Tom Lampert, Field Applications Scientist

Thursday, November 15, 2018

Neuroscience 2018 - Making Connections: From Cajal to the Connectome

When brain cells are wired in a manner that strays from their normal connectivity, functional consequences arise. Abnormal neuronal morphology and connectivity has implications in a wide variety of brain diseases and disorders from neurodevelopmental disorders to cognitive ageing. Collectively known as the connectome, researchers have been mapping neuronal connections in the brain for almost 150 years. Since the days of Neuroanatomist Santiago Ramón y Cajal, neuroscientists have been drawing and mapping the structure of neurons and other brain cells in an attempt to elucidate their function and understand how the brain works. Little did Cajal know back in 1906, that his methods of archiving neurons would change very little into the 21st century. Despite advances in techniques to label neurons, and although new computational methods to draw their morphology have replaced the Golgi stain and the paper and ink Cajal used, not much else has changed. His work is still very much relevant today and neuroscientists still draw each neuron manually, albeit digitally. Interestingly in this age of bioinformatics, the field is still struggling to find a way to archive and connect the input/output trees of all of the neurons researchers have drawn by hand in software packages such as Neurolucida or ImageJ/Fiji.

Santiago Ramón y Cajal, Golgi stained pyramidal cells of the cerebral cortex (detail) ink and pencil on paper.
 Courtesy of the Cajal Institute, Spanish National Research Council, or CSIC. Madrid. Spain.
The process of labeling neuronal nuclei is straightforward, inexpensive and accomplished in a variety of ways. Neuronal nuclei in a brain tissue section or in a 2D neuronal culture are visualized using fluorescence markers or colorimetric detection such as DAB and Nissl staining. With the exception of slight size differences, all neuronal nuclei are relatively similar in morphology.

Fig 1: Left- H&E stained sagittal mouse brain tissue section, montaged, stitched
4x on the Cytation™ 5, right-inset, digitally zoomed hippocampal region
Only when you begin labeling the processes of these cells, do their differences in structure begin to unfold. Furthermore, because of the sheer number of connections in what appears to be a tangled web of dendrite and axonal networks, untangling these connections to make sense of this wiring diagram introduces an enormous amount of difficulty and complexity. From images like these (Fig. 2) however, researchers can screen their targets in a high throughput manner using different HTS imaging platforms, without the need for laborious, manual tracing. Assays like these provide readouts for metrics like global neurite outgrowth, synaptogenesis, differentiation and phenotypic analysis such as the one presented at Neuroscience 2018 by C. Pandanaboina et al.

Fig 2: primary culture of neurons and astrocytes at 20x on the Cytation™ 5 labelled with
MAP2 (red) and GFAP (green) respectively and nuclei counterstained with Hoechst.
However, there will always be a need to dissect brain circuitry to parse out different aspects of development and disease. Likewise, in order to accomplish this, open access to findings will be key in understanding complex brain networks. Researchers in a wide variety of neuroscience disciplines are finally coming together in a concerted effort to build a central resource to assess these complexities using computer models and machine learning algorithms. Ten years ago, Giorgio Ascoli and researchers at Krasnow Institute at Georgetown University launched a digital repository for anyone who created neuron tracings in any species or region of the brain to create a database of sharable, downloadable content. It is one of the only digital neuronal reconstruction databases to date. Giorgio presented some of the challenges and accomplishments of their work recently at the Neuroscience 2018 meeting in San Diego. These methods are still quite labor intensive and expensive. From the nearly 100,000 neuron trees in the database, it has taken researchers over 100,000 years cumulatively and $2.5 million dollars in research funding.
Fig 3: Human, male, neocortical, pyramidal neuron from the database.
NeuroMorpho is currently collaborating with BigNeuron project, from the Allen Institute for Brain Science, and is another potential resource. Along with several other online 3D reconstruction resources such as MatLab (MathWorks), Vaa3D and Imaris (Bitplane), users can upload their tracings to the NeuroMorpho.Org database. This will help the community build a curated neuronal network across species and brain regions in hopes of connecting morphologies to subtle functional consequences.

  1. Ascoli, G. A., Maraver, P., Nanda, S., Polavaram, S., & Armañanzas, R. (2017). Win–win data sharing in neuroscience. Nature Methods, 14, 112. Retrieved from 
  2. Akram, M. A., Nanda, S., Maraver, P., Armananzas, R., & Ascoli, G. A. (2018). An open repository for single-cell reconstructions of the brain forest. Scientific Data, 5, 180006.
Further inquiry and reading resources: 
  1. Meijering, E. (2010), Neuron tracing in perspective. Cytometry, 77A: 693-704. doi:10.1002/cyto.a.20895 

By: BioTek Instruments, Sarah Guadiana, Ph.D, Field Applications Scientist, Imaging

Tuesday, October 23, 2018

There’s No Place Like Home, Even (and Especially) for a Cell

Traditional monolayer cell culture in plastic dishes has been the standard for many assays and high throughput screens for decades, in large part due to its ease and low cost. Unfortunately, established cell lines grown on flat, plastic surfaces cannot truly recapitulate the phenotypes, signaling, and microenvironment of their native tissue, leading many to question the relevance of scientific conclusions drawn from monolayer assays. In response, cell culture methods are increasingly moving toward improving our recreation of a cell’s “home” environment [1].

Cells in their native tissue are surrounded by other cells and a complex web of proteins that provide a tissue-specific stiffness optimized by evolutionary pressure over millennia. Cells are bathed in a biochemical cocktail of carefully balanced signaling molecules and nutrients that dynamically changes in response to the status and needs of the overall tissue. This microenvironment drives the cell to adopt a specific morphology and perform a specific task in the context of the cells and signals around it, ultimately cooperatively regulating complex biological functions. This is the environment that carefully directs development, balances health and disease, and dictates response to drugs.

By contrast, cells grown on tissue culture plastic are forced to adapt to growth against a rigid structure on one side and bathed in liquid media on the other. In this setting, most cells adopt a flat, strained morphology against the stiffness of the plastic, are less able to form traditional cell-cell junctions, and, as all cells in the culture are equally exposed to the same media for days, there is no dynamic biochemical microenvironment to direct specificity in phenotype or function. Everything from gene expression to proliferation to metabolism and drug sensitivity have been shown to be altered by monolayer culture as compared with in vivo measurements.

So how do we encourage cultured cells to behave as they do in the body? Three dimensional (3D) cell culture, pioneered in the 1980s, has matured over the past 3 decades to provide both manual and automated tools for accurate artificial modeling of physiologically relevant structures that can be adapted for both highly intricate low throughput investigations and improved accuracy for high throughput drug screening. 3D methods, though not perfect, more accurately reproduce in vivo physiological conditions resulting in cells that respond to experimental stimuli with better correlation to clinical expectations. 3D culture methods can be grouped into 3 categories of differing complexity and physiological relevance:

SUSPENSION CULTURE: The simplest design for creating a 3D cell culture model is to plate the cells in a low-adherence environment or hanging-drop assay and allow them to self-assemble into aggregates. Cells cultured in this way will secrete their own extracellular matrix and form appropriate cell-cell junctions and interactions. Aggregates may be tight (to form spheroids) or loose (looking instead like clusters) depending upon the characteristics and needs of the cells themselves.
Benefits: Suspension culture can maintain the ease and low cost of traditional monolayer culture, making it suitable and scalable for high throughput applications.
Drawbacks: Cells in aggregates will not fully model the desired tissue, but can mimic some aspects of the cell’s home environment. Additionally, cell aggregates are floating and thus for imaging applications may pose some obstacles with excess movement and high variability of their focal plane.
When using a BioTek Lionheart or Cytation imager, increase the delay after stage movement to account for the floating aggregates and try acquiring a z-stack with a large step size in order to capture a broader depth of cells.
Lionheart FX
Lionheart FX Automated Microscope
  • SCAFFOLD CULTURE: Hydrogels, electrospun membranes, bioprinted scaffolds, or other matrices can be artificially engineered to provide additional structure and biochemical recapitulation of the natural tissue environment. Cells can be cultured on or embedded within the matrix, which can also be loaded with nutrients, growth factors, drugs, or other signaling compounds, depending upon the needs of the assay or the requirements of the cell type.
    Benefits: Physical characteristics of the scaffold can be optimized to more accurately model the cell’s native influences. By promoting appropriate stiffness, cell-cell interactions, and cell-matrix interactions, scaffold-based 3D cell culture can drive organotypic gene expression and cellular organization that more accurately mimics activity in the body.
    Drawbacks: Scaffold or matrix-based 3D culture can be costly in both time and money, making it less desirable for high throughput applications, though not impossible. Preparing the matrix/scaffold can be technically demanding (even some commercially available hydrogel-based matrices can be difficult to manipulate) which can result in poor structure formation and/or difficult imaging conditions.
    When working with hydrogel-based matrices for 3D culture, lay down 90% of the total volume and allow it to gel before adding the final 10%. This will result in a more consistent gel layer, which will result in improved imaging conditions and uniformity of 3D structures. In Gen5, by optimizing the autofocus parameters in Experiment Mode, you can easily maintain reliable focus during automated acquisition of scaffold-based 3D assays. 
  • ORGAN ON A CHIP: One of the newest and most intriguing applications of 3D cell culture is the development of Organ on a Chip, which takes 3D cell culture to the next level, utilizing co-culture and microfluidic chambers to fully mimic in vivo functionality as well as model the interfaces between tissues [2]. This approach has successfully simulated heart, lung, kidney, skin, and bone, and it is hoped that this technology may one day reduce or eliminate the need for animal studies.
    Benefits: Organ on a Chip is the in vitro method able to most accurately model multiple organ systems as well as the dynamic balance between health and disease.
    Drawbacks: Organ on a Chip is still in early stages of development for many organ systems and is not yet scalable to mid- or high throughput. Much work remains to be done on this technology.
    Custom labware can be created within Gen5 to create specific Plate Definitions for Organ on a Chip or any other unique cell culture vessel. Simply use a picture of the vessel in the appropriate stage adapter (such as the multi-vessel adapter for Organ on a Chip) to drag and drop imaging regions where needed.
  1. Ye Fang, Richard M. Eglan. Three Dimensional Cell Cultures in Drug Discovery and Development. SLAS Discovery, 2017. DOI: 10.1177/1087057117696795 
  2. Dongeun Huh, Benjamin D. Matthews, Akiko Mammoto, Martin Montoya-Zavala, Hong Yuan Hsin, Donald E. Ingber. Reconstituting Organ-Level Lung Functions on a Chip. Science, 2010. DOI: 10.1126/science.1188302 

By: BioTek Instruments, Diane M. Kambach, FAS Team Leader

Thursday, October 18, 2018

Simplifying Spheroid Media Exchange using the AMX Automated Media Exchange Module

3D cell culture methods encourage cell-cell and cell-matrix interactions and also promote more physiological cell morphologies and behaviors compared to cells grown on flat 2D surfaces. Due to the need to generate the most in vivo like data during in vitro drug discovery or toxicology testing, 3D spheroids are often used in long-term experimental procedures, and in microplate format to increase throughput. Since these procedures may extend to weeks, media exchange and dosing steps are critically important. When unattached spheroids are incorporated, performing aspiration and dispense steps manually can be slow and painstaking, and can still result in lost, damaged, or non-imaged spheroids

To solve these complications and make 3D assay procedures easier to perform, BioTek has developed an Automated Media Exchange module (AMX) to perform single media exchanges or multiple plate washes in a slow, controlled manner that allows liquid to be aspirated and dispensed from the wells of a spheroid microplate without removing or disturbing spheroids. The module consists of two unique, peristaltic pump cassettes each with a manifold containing eight stainless steel tubes. Cassette tubing is fed through the peristaltic pumps of BioTek’s MultiFlo™ FX Multi-Mode Dispenser and into bottles or tubes containing media. Software controls the pumps to run slowly and gently so as to not disturb the spheroids. The secondary pump is run backwards to remove liquid, while the primary pump is run in a forward direction to dispense liquid back to each well.

Automated Media Exchange Procedure. (Left) Liquid aspiration; and (Right) dispense by AMX cassette tubes.

By positioning the tubes at the back corner of the well and slightly elevated from the well bottom, away from the spheroid (see above figures), 80-90% of the liquid within the well can be removed and replaced with an appropriate volume for the procedure being completed.

Currently, validated methods exist for 96- and 384-well plates from a variety of plate vendors, and are available upon request.

To find out more about the AMX Automated Media Exchange module, we invite you to visit the following webpage:

By: BioTek Instruments, Brad Larson, Principal Scientist

Wednesday, October 3, 2018

A new Superpower in the Quest for Healing Medicines

Our family recently grew in size… we got a ridiculously adorable puppy that has been keeping us quite busy, both day and night. Truth be told, he is already doing great on most of the typical puppy problem areas – learning to do “his business” outside, sleeping through the night, crate training, etc. The one area where he is still displaying 100% of his playful puppy nature is his chewing and biting. All four sets of hands in our house have punctures and wounds from his teeny-tiny, razor sharp puppy teeth. As I stare down at my hands, it makes me wish there were better medicines out there for quickly healing all my scratches and bites.

In fact, there is a large field of research tied to this exact area - healing wounds - and, the inverse, keeping cells from migrating. One of the leaders in funding wound healing research is the US Department of Defense. They have a keen interest in finding medicines and drugs to improve healing wounds, which would aid dramatically in medicine for warfare. On the other side, the field of oncology is interested in drugs that keep cells from migrating. Drugs with that behavior, specifically targeted at cancer cells, could help treat cancer metastasis and growth. With any research field, scientists work hard to keep all variables in an experiment as standardized and repeatable as possible. In the area of wound healing, one of the key challenges has been standardizing the size and shape of scratches through a layer of cells, to mimic the look and feel of an actual wound. Many researchers today use standard polypropylene pipette tips to create scratches. These do the job, however the scratches through cell monolayers are quite variable. This can create a huge challenge in measurements since the scratches are highly variable in size and shape, which can lead to a significant amount of noise in the final data.

This is where BioTek’s new AutoScratch Wound Making accessory shines (in my mind I have epic music playing while it flies in, stage left). AutoScratch has one purpose in life – to make beautiful, repeatable, make-scientists-smile-with-delight, 800 micron wide scratches through a cell monolayer in either a 24 or 96 well Corning Costar microplate. The innovativeness of the design means that everything is automated, thus removing all the variability that comes from a user making their own scratches. And, for those scientists who hate cleaning up after themselves (I am one of them)... even the cleaning routine is automated! You just can’t beat that!

Automated wound creation using AutoScratch
Manual wound creation using a pipette tip

After AutoScratch exits, stage right, all that is left for the researcher to do is to add drugs looking for either inhibition or activation of wound healing. At the completion of the assay, BioTek offers a number of intuitive imaging systems that will automate the image capture of all the scratches, and will measure key metrics such as the wound width, wound confluence, and max acceleration rate of the wound closure. As I type these last few sentences - with my battered, puppy-scarred hands - my hope is that one of you reading this will have a moment of enlightenment: "Hey, I could use the AutoScratch to discover new wound healing medicines for people just like him". Maybe, just maybe, the next time my family adopts a puppy, there will be a new medicine on the market that heals puppy bites - all enabled by the AutoScratch, this blog, and your ingenious scientific spirit. Please - my hands beg you - don’t delay!

For more information on AutoScratch click here.

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

Tuesday, August 28, 2018

A Matter of Time

When evaluating treatment-induced effects on cells, timing is critical. Cells respond to changes in their environment in diverse ways – from sub-second signaling cascades to changes in cell health that accumulate over days to weeks. The ability to characterize events occurring over these markedly different timescales provides unique insight into cellular processes and increased flexibility for drug development studies.

End-point assays generate a single snapshot of these processes – often at an arbitrarily chosen time point – that is incomplete and easy to misinterpret. In contrast, kinetic imaging-based assays provide a detailed profile of cell characteristics over time, enabling unique quantitative analysis and intuitive validation of results.

BioTek Instrument’s versatile automated imaging systems allow researchers to monitor live cells over a full range of time courses, from seconds to weeks. The instruments maintain optimal environmental growth conditions for long-term studies of cell stress and viability, while an image capture rate of up to 20 frames per second, and aligned dual reagent injectors, enable characterization of rapid cell signaling events.

An example of the unique experiments that are possible with BioTek’s Cytation™ or Lionheart™ FX imaging systems comes from a recent study using expressed biosensors from Montana Molecular. Chemically-induced cell stress by thapsigargin, a potent SERCA pump inhibitor, was measured over a 24 hour period using a new cell stress sensor, while effects on Gq-mediated cell signaling where simultaneously monitored with the R-GECO calcium sensor.

Monitoring stress over 24 hour period in HEK293 cells treated with 1 µM thapsigargin.

These results reveal that cell stress induced by thapsigargin is detected at an order of magnitude lower concentration compared to discernible effects on cellular proliferation. Stress levels peak after 6 hours of treatment. After which, cells either recover naturally from this stress within 22 hours (e.g. 0.1 and 0.3 µM thapsigargin) or progress to more advanced states of distress (i.e. mitotic arrest or cell death).

The effect of thapsigargin on Gq-dependent Ca2+ signaling was probed by adding hM1 receptor agonist to treated cells using the reagent injectors. Ca2+ signaling was reduced in a dose-dependent manner at both 6 and 22 hours during thapsigargin treatment, indicating that the effects of ER stress on Gq-mediated Ca2+ signaling remain even after the cell has shut down the stress response.

Monitoring Gq-dependent calcium signaling in HEK293 cells 6 hours after 1 nM thapsigargin treatment

Click on the image to see larger version.

The ability to monitor cell stress and associated effects on cell signaling is important for understanding a broad range of diseases, as well as for the process of developing drugs designed to treat them. The unique combination of kinetic assays that are possible with BioTek automated imaging systems provides researchers with more physiologically relevant insight and expands research possibilities.

Visit our website to learn more about the full range of application solutions available with BioTek Instruments.

By: BioTek Instruments, Joe Clayton, PhD., Principal Scientist