A recent report indicates that circa 79% of 483 warning letters issued by the FDA to the biopharmaceutical industry in 2016 cited deficiencies in data integrity. The FDA outlines their expectations for quality critical instrumentation in the GMP environment in their 21 CFR Part 11 ruling. This webinar takes a look at how quality-critical on-line Total Organic Carbon (TOC) and conductivity instrumentation can be configured to help companies comply with the FDA’s expectations on data integrity, including the use of Microsoft Active Directory and PDF data export.

Counterflow centrifugal elutriation (CCE) is a well-known technique used commonly several decades ago in scientific research. In this technique, cells are subjected to two opposing forces, centrifugal force and fluid velocity, which results in the separation of cells on the basis of size and density. With the advent of more sophisticated and automated technologies, the technique of CCE has taken a back-seat. However, CCE can still provide unique knowledge and scientific breakthroughs in research in several areas. There is a need to revive this technology in order to benefit the most from its unique capabilities. This webinar is aimed at describing the technique of CCE and how a wide range of scientific areas can apply this tool to advance their investigations. This technique is especially helpful in studying aspects of primary acute lymphoblastic leukemia (ALL) cells. Under optimal conditions an essentially pure population of cells in G1 phase and a highly enriched population of cells in S and G2/M phases of cell cycle can be obtained in excellent yield. These cell populations are ideally suited for studying cell cycle-dependent mechanisms of action of anticancer drugs and for other applications. The limitations of the technique will also be discussed. The detailed methodology presented should facilitate application and exploration of the technique to other types of cells.
 

According to a recent report, 79% of FDA 483 Warning Letters issued in 2016 cited data integrity. In their guidance on data integrity and compliance with CGMP, the FDA issued their ALCOA definition of data integrity. This seminar will discuss ALCOA using cleanroom environmental monitoring data collection in the pharmaceutical industry as an example.

As gene therapy approaches usually require large amounts of AAV vectors for clinical use, few manufacturing processes have been reported to provide high titer and potent quantities of products.
The dose control of AAV vectors is commonly established on titration methods relying either on the quantitation of DNA (viral genome) or transgene expression following cell transduction (infectious genome). However, AAV vector lots are generally a heterogeneous mixture of empty particles (i.e. do not contain DNA) and full particles (i.e. contain DNA). Therefore, it could be considered that empty particles are product related impurities, which can impact on the immunogenicity profile of the product when high doses are administered to patients.

Different indirect methods can be used to establish the ratio between full and empty AAV particles. The quantification of full capsids is performed by using qPCR based technology. The total particles can be evaluated by an ELISA assay, spectrometric analysis, ion-exchange chromatography or SDS-PAGE. However, these methods have limitations and are not applicable to all serotypes without performing a new development.

Using analytical ultracentrifugation approach, we have developed a method to quantify simultaneously empty and full AAV particles as well as intermediate species containing fragmented or incomplete vector genome. We have applied this technique to AAV lots of different serotypes, several sizes of transgene and different process of production. Several examples of AAV vectors analysis will be presented showing that AUC could be implemented as a routine test to monitor AAV product quality and manufacturing consistency.

Cell culture and cellular applications have been and continue to be essential to medical and biological research. By automating these assays, researchers can reduce their time at the bench while processing more samples to accelerate discoveries. We will demonstrate how automation technologies from Beckman Coulter can be used for a variety of cellular applications including basic cell culture and maintenance, transfection optimization, flow cytometry assays, and ELISAs. We will also discuss how to automate sample processing over multiple days – a common occurrence in cell culture – as well as how data can be tracked and used to drive additional liquid handling steps during the process.

Please note this is the third webinar in a three-part series. If it’s not too late, make sure to register for the first two so you can attend live or download an on-demand version.

• The Future of Liquid Handling Automation has Arrived on Wednesday, March 22 at 10:30 a.m. ET/7:30 a.m. PT 
• Don’t hesitate. Learn how to step up your library prep with new Biomek i-Series Genomic Sample Prep Workstations on Tuesday, April 18 at 10:30 a.m. ET/7:30 a.m. PT  

Labs large and small that conduct genomic studies are looking for ways to vastly improve NGS sample prep, especially from challenging FFPE and low-input samples. This educational webinar will detail a Biomek i-Series automated workflow solution for biomarker discovery,  starting with DNA extraction from FFPE to exome enrichment through NGS results.  You will learn how the new Biomek i- Series Automated Workstations can help labs increase productivity, efficiency, and turnaround time without compromising results. Join us to learn how to accelerate your research with less hands-on time.

Please note this is the second webinar in a three-part series. Make sure you register for: 

• The Future of Liquid Handling Automation has Arrived on Wednesday, March 22 at 10:30 a.m. ET/7:30 a.m. PT
• Advances in Cellular Automation on Thursday, April 20 at 10:30 a.m. ET/7:30 a.m. PT

This webinar provides an overview on laser diffraction analysis as a tool to characterize particle size distributions in the field of geoscience and soil science, highlighting its capabilities, significance and potential to obtain improved results and novel scientific findings. It describes methods of optimized and standardized sample collection, preparation and measurement as well as preferable technical peripheral appliances and set-up. For emphasis, selected case studies are presented, further illustrating that a high measurement resolution and high number of detector elements - characteristic features of the Beckman Coulter LS13320 PIDS laser diffraction analyzer - are key for applications in the wide field of geoscience and soil science.

Learning Objectives:

• Laser diffraction analysis as a tool to characterize particle size distributions in the field of geoscience and soil science
• Preparation and treatment of geoscience materials for laser diffraction measurements
• How particle size distributions in sedimentary sequences and soils are crucial for research on sedimentary dynamics, paleoenvironmental and climate evolution, and landscape development

Contamination is the #1 cause of hydraulic component failure, being responsible for an estimated 60% to 75% of all problems in a typical hydraulic system.

Learn how contamination affects hydraulic components, how much you can save by controlling
contamination, and 3 ways to deal with contamination in your hydraulic systems.

Learning Objectives:

• Learn how the #1 cause of hydraulic component failure affects hydraulic components.
• Learn how to extend hydraulic component life and reduce down time.
• Learn the requirements for hydraulic fluid cleanliness.

Un-analyzed data isn't useful.  Poorly analyzed data is useless.  And that means no funding!  But how to overcome the analysis burden without a team of biostatisticians at your disposal?  Kaluza can help!  At our contract research organization, time is money and potentially an impediment to drug delivery.  We have designed templates with advanced analytical tools to streamline analysis and report generation to address our clients 'need for speed' and the need to operate in a GLP (Good Laboratory Practice) fashion.

Higher throughput analysis with statistics reporting on board:

1. Learn to maximize data output utilizing composites
2. Generate reports while retaining the raw data in the same file
3. Export to excel not required!
4. Large files easily manipulated
5. Link gates across study samples

Kaluza Analysis Software is for research only and should not be used for diagnostic purposes.

Increasingly, Formalin-Fixed, Paraffin-Embedded (FFPE) samples are an essential part of many next-generation sequencing (NGS) sample prep workflows, specifically in the field of cancer research. High quality extraction protocols focused on yield and purity are essential to achieving reliable results. FormaPure DNA now provides an automation-ready, SPRI-based extraction reagent kit to support your evolving research needs.
                                                                                 
Most notably, it provides significant time savings in the form of reduced turnaround times and less than 15 minutes of hands-on time when automated on a Biomek liquid handler. Additional benefits include:

• Dramatically increase productivity through automation - low to high throughput
• Proven compatibility with NGS and other downstream applications
• Consistent extraction performance
• Enhanced lab safety

Webinar attendees will learn about:

• Situations when using FFPE samples for NGS is a better option than fresh-frozen samples
• The challenges of extracting DNA from FFPE samples manually
• A demonstrated automated extraction solution that maintains sample quality and yield while significantly reducing turnaround and hands-on times

Does your protein analysis toolbox need a refresh? Do you have a firm grasp on the tools available and when each might best be employed in your daily research activities?
 
High quality proteins are critical for contemporary biomedical research. Biochemists and biophysicists require high quality proteins to probe the molecular mechanisms of proteins in their native state. Proteomics researchers, cell biologists, and pathologists require high-quality antibodies as purification tools and as diagnostic reagents. Formulation scientists need to quickly gain an understanding of the impact that buffer and other components have on the structure and function of protein biologics.
 
In this webinar, several complementary protein analysis tools will be introduced and discussed including chromatography, UV/VIS spectroscopy, light scattering, electrophoresis, western blotting and analytical ultracentrifugation.  In closing, I will discuss our work in measuring the pairwise interaction matrices of hundreds of ErbB signaling proteins and peptides to illustrate the importance of quality control in obtaining high quality protein interaction data.
 
Learning Objectives:

• Learn about approaches for the purification and quality analysis of proteins and antibodies
• Learn about systematic methods for assessing interaction potentials of proteins 
• Learn about higher-throughput western blotting methods

Clinical laboratories are under increasing pressure from three sources: reimbursement, regulatory, and resource. Reimbursement is the most pressing issue for many labs: whether in a fully-bundled, capitated environment or in a fee-for-service setting, laboratories are being asked to decrease their operating expenses. Key to doing so is understanding the relative contributions of labor and consumables. Laboratory-developed tests are particularly labor-intense, with labor accounting for over 70% in many instances.  Increasingly rigorous regulatory requirements are another concern, particularly in geographies where laboratory-developed tests are under scrutiny.  Meeting regulatory requirements will place demands on both time and expertise. Resource, specifically human resource, is the third challenge for clinical laboratories. The medical technologist labor pool is simultaneously shrinking and aging, and competition for this increasingly scarce resource contributes to the overall cost of laboratory operation. Training, particularly in laboratories that perform high complexity, laboratory-developed tests, is an additional cost that affects both bottom line and productivity.
 
In this webinar we will:

• Describe the three sources of pressure, their underlying drivers, and the ways in which they’re interconnected
• Discuss the “8 Wastes” in the context of the laboratory
• Provide tips for assessing impact of waste
• Show examples of streamlined workflows that emphasize both efficiency and quality patient care

The FDA’s 21 CFR Part 11 guidance on the use of electronic records for data retention or submission is clear. With manually implemented Standard Operating Procedures for Quality Control testing and manual transcription of the test results still common practices in the pharmaceutical QC world, the resultant opportunities for human error raises concerns over the integrity of the data in the final electronic record, no matter how safely the record is stored.

This Webinar describes how Quality Control instruments can be optimised for pharmaceutical use, helping to improve the integrity of the data in electronic records.

Enzyme engineering is a powerful technology now widely used in laboratories around the world. The goal is to obtain improved proteins that will serve as better biocatalysts, biosensors or as a tool to understand protein evolution, which is at the core of many societal problem, such as antibiotic resistance. Enzyme engineering entails the initial generation of libraries of mutants that will then be screened for improved properties (for example: better selectivity or specificity towards a substrate of interest, improved stability, etc). Obtaining good quality libraries is becoming routine thanks to the availability of molecular biology tools and computational simulations. The bottleneck of the experiment remains at the screening level. The bigger the generated library, the more stringent the requirement for the high-throughput screen. 
In our work we tackle both aspects: library generation and screening throughput by using the enzyme Candida antarctica lipase A (Cal-A) as a model. Cal-A has the potential to be applied in the food industry for the hydrolysis of saturated short-chain fatty acids (C4 and C6) vs. saturated C10-C16 fatty acids - a very useful tool for the dairy industry. Upon generation of several libraries of mutants using a novel Golden Gate-based strategy, we developed two screening methods (in vivo and in vitro) that give us the required high-throughput capacity. The two screening methods, one carried out on agar plates and the other in liquid, are completely automated and they are both optimized for our Biomek NX^P system.

July 13, 2016, 8:00am PT, 11:00am ET, 4:00pm GMT
 

Changes in the classification standard, ISO 14644-1:2015, will have an impact on both the number of sample points needed to classify and on the quality and maintenance of the instruments needed to comply. The impact of the latest revision affects all cleanrooms users, even if you classify following Annex 1 from the EU GMP or PIC/s. It is also relevant to particle counters used in the EM program for routine monitoring or investigations.

This webinar focuses on the key changes on the ISO 14644-1 standard with an emphasis on understanding the role of the calibration standard, ISO 21501-4, referenced in the latest revision.

Make sure your methods and equipment for classification will comply!

Learning Objectives:

• Understand the key changes on the ISO 14644-1 standard with an emphasis on understanding the role of the calibration standard, ISO 21501-4, referenced in the latest revision.
• Learn if your methods and equipment for classification will comply

Precision medicine requires success in two intertwined aspects: precision therapy and personalized medicine. Precision therapy is being able to effectively treat the right disease; to have therapies that target for the correct pathological pathways.  Personalized medicine requires diagnosing a specific individual’s disease based on accurate assessment their complex health and pathological status. Our underlying premise is that an individual’s baseline proteome reflects their past and present and thus, will dictate their future health and disease. Thus, the crux of precision medicine will be the identification and precise quantification of proteins and their modified forms. We will present data to support the notion that an individual’s baseline proteome dictates the manifestation of their disease, its progression and their response to therapy. This concept has driven decision-making processes around new drug trials that take into account altered pathological proteomes.  Furthermore, this work has led us to consider the need for continuous patent-centric biomarker analysis at the population level. We will share our work on developing technical pipelines for health and pathological discovery and targeted proteomic analysis that reduces the barriers around gender, age and social economic status. We have begun down this path with production of system suitability and quality control measures, development of quantitative small and large multiplex assays and will discuss the remaining challenges involved and requirement for population-scale proteomics.

Learning Objectives:

• Understand the underlying premise that an individual’s baseline proteome reflects their past and present and thus, will dictate their future health and disease
• See data to support the notion that an individual’s baseline proteome dictates the manifestation of their disease, its progression and their response to therapy

April 14, 2016, 7:00am PT, 10:00am ET, 2:00pm GMT

Whether cells are being used to manufacture a therapeutic drug product or for pharmacology, measuring the cell viability and concentration accurately and with good reproducibility is crucial. This presentation describes how to avoid the common pitfalls when adopting a new, automated method of cell counting.

Learning Objectives:

• Learn key critical elements of automated cell viability counter adjustment for correct cell counting
• Learn key pitfalls when automating cell viability counting

Protein aggregates can impact patient safety. This is especially important in the Biotech industry. Regulatory agencies are very worried about protein aggregates. Dr. Stoner breaks his webinar into three parts. He first starts with a discussion of the AUC itself and protein aggregates. He talks about how we use AUC to quantify protein aggregates. Next Dr. Stoner talks about how size exclusion chromatography compares with sedimentation velocity. Lastly, he shows a simulation and describes how it’s a very powerful tool to see what you can get out of the AUC and to be able to understand the results that you’re getting.

Learning Objectives:

• Understand how size exclusion chromatography compares to sedimentation velocity
• Understand how the Analytical Ultracentrifuge (AUC) is used to quantify protein aggregates
• See how The Analytical Ultracentrifuge (AUC) is a powerful tool and understand the results you can get from the instrument

February 25, 2016, 9:00am PT, 12:00pm ET, 5:00pm GMT

This webinar, presented by one of the US members to the international revision group, discusses how the newly-revised ISO 14644-1 and -2 standard for counting airborne particles in cleanrooms may impact your classification and monitoring procedures. Classification is now discussed solely in the ISO 14644-1 section, detailing the methods to conduct the formal classification effort. In many cases, the revision requires an increase in the minimum number of locations needed for classification. The concept of the selection of positions as “representative” or “risk-based” are presented for the first time. The companion ISO 14644-2 section focuses on the design and characteristics of a monitoring strategy to support continued effective operation of a cleanroom or clean zone.

This webinar will be helpful to all users of cleanrooms or clean zones (such as isolators, gloveboxes, BSC or filtered flow benches) and will provide useful information for plant managers, microbiologists, QA and QC managers as well as the persons actually executing the testing for airborne particles. All industries operating cleanrooms or clean zones will be impacted by this revision but additional commentary will be made on the relationship of these ISO documents and key biopharmaceutical documents such as the EU/PIC/s Annex 1. There will be a Q&A session at the end of the presentation.

Learning Objectives:

• Learn how the newly-revised ISO 14644-1 and -2 standard for counting airborne particles in cleanrooms may impact your classification and monitoring procedures
• Learn useful information for plant managers, microbiologists, QA and QC managers as well as the persons actually executing the testing for airborne particles. 

January 28, 2016, 8:00am PT, 11:00am ET, 3:00pm GMT

The relationship of particle size distribution (PSD) in cement and its impact on both strength and curing time of concrete make PSD critical to maintaining consistent product quality. Historically, sieves were the preferred method for determining PSD in cement. However, over the last 20 years, use of laser diffraction instruments have evolved into the preferred method for determining the distribution of particles in cement. This webinar will focus on unique features in the Beckman Coulter LS 13 320 that simplify sample preparation, decrease analysis time and improve accuracy. Accuracy data will be compared to NIST traceable SRM 114q to demonstrate the resolution of the Beckman Coulter LS 13 320.

Learning Objectives:

• Understand how use of laser diffraction instruments have evolved into the preferred method for determining the distribution of particles in cement
• Understand unique features on the Beckman Coulter LS 13 320 laser diffraction particle size analyzer that simplify sample preparation, decrease analysis time and improve accuracy 

 

January 12, 2016, 8:00am PT, 11:00am ET, 3:00pm GMT

Targeted nanoparticle applications have become a major focus of translational development of both diagnostics and therapeutics for clinical use.  One of the oldest and best established nanoparticle formulations is liposomes, which can be readily targeted to tissue markers by chemical conjugation to ligands such as antibodies.  The cardiovascular research group at UTHealth has been exploring molecular targeting strategies for our intrinsically echogenic liposomes (ELIP) for nearly 25 years.  As part of this effort, we have developed evaluation methods for both conjugation and targeting efficiencies that rely on particle characterization techniques, especially the Beckman Coulter Multisizer.  This webinar will present the immunoblot assay (IBA) that measures IgG concentrations in the presence of liposomes.  We use the Multisizer to enumerate population based on size. We will present several sophisticated methods developed by us for determining targeting efficiency (TE), based on the CE and ELISA-measured antibody (Ab) binding affinity and binding efficiency.  The most informative measure of TE, however, is the specific TE, which is the product of the number of Ab molecules that actually bind to immobilized target molecules (determined by comparing the ELISA CE to the IBA CE) and the affinity.

Learning Objectives:

• Learn about the immunoblot assay (IBA) that measures immunoglobulin G (IgG) concentrations in the presence of liposomes.
• Learn about sophisticated methods developed for determining targeting efficiency (TE), based on the CE and ELISA-measured antibody (Ab) binding affinity and binding efficiency 

Innate Lymphoid Cells (ILC) are lymphocytes that share the same morphological features of classical lymphocytes. They lack specific antigen receptors (Spits and Cupedo Annu Rev Immunol 2012). The ILC family contains Natural Killer cells (NK), lymphoid tissue inducer (LTi) and other ILCs (Spits et al. Nat Rev Immunol 2013). A classification was proposed in which they divided the ILC family into three different groups based on their capacity to secrete cytokines (Spits et al. Nat Rev Immunol 2013). ILC group 1 is able to secrete IFN-g, ILC group 2 is able to secrete type 2 cytokines (IL5 and IL13) and ILC group 3 is able to secrete IL17 and/or IL22. ILC could also be classified by their expression of different membrane receptors. According to Mette and collaborators, the ILC are lineage negative, CD127+, and CD161+. The classification is made on CD117, CRTh2 and NKp44. ILC1 are CD117- CRTh2- and NKp44-. ILC2 are CD117+/- and CRTh2+. ILC3 are CD117+ and NKp44+/- (Mette et al Blood 2014).  In this presentation, we will propose a method to phenotype whole blood ILCs and PBMC ILCs. We will present how to use the new CytoFLEX flow cytometer to look into these different human classical ILC. We will also see how to calculate automatic compensations, and how to set carefully the different gates using fluorescence minus one tubes.

Learning Objectives:

• See a proposed method to phenotype whole blood ILCs and PBMC ILCs
• Learn how to use the new CytoFLEX flow cytometer to look into these different human classical ILC
• Learn how to calculate automatic compensations, and how to set carefully the different gates using fluorescence minus one tubes using the CytoFLEX flow cytometer 

Cancer-derived extracellular vesicles (EVs) play an important role in cancer progression and metastasis. They can be identified in biological fluids thus providing appealing candidates for novel circulating biomarkers. Studies designed to achieve a deeper understanding of the extent to which EVs propagate oncogenic signals and can be interrogated in clinically relevant settings are increasing. EVs are highly heterogeneous in size, function, biogenesis and mechanisms of communication with target cells. A greater molecular and functional characterization of the EV types that can be released from a given cancer cell will improve our knowledge of the mechanisms underlying cancer progression. Focus of this webinar is a novel EV population referred to as large oncosomes, which originate from the shedding of non-apoptotic membrane blebbing. This biological phenomenon is typical of amoeboid tumor cells with keen migratory abilities and high metastatic propensity. Recent studies demonstrate that large oncosomes are molecular and functional entities that can be distinguished from exosomes and possibly from other EV classes. Further studies on the functional role of large oncosomes and other EVs in specific steps of cancer formation and progression will expand our understanding of the diversity of paracrine signaling mechanisms in malignant growth and metastasis.

Learning Objectives:

• Understand importance of the role cancer-derived extracellular vesicles (EVs) play in cancer progression and metastasis
• Understand the role of novel EV population referred to as large oncosomes
• Understand a novel extracellular vesicle (EV) population referred to as large oncosomes, which originate from the shedding of non-apoptotic membrane blebbing
• Understand recent studies that demonstrate that large oncosomes are molecular and functional entities that can be distinguished from exosomes and possibly from other EV classes

In this webinar, you will learn about an innovative approach to a traditional technology which encompasses several advanced features that are unusual for a flow cytometer.  For example, the CytoFLEX includes a communications technology called Wavelength Division Multiplier (WDM) that changes the paradigm of light collection techniques in flow cytometry.  This technology is also transformational in that it integrates APDs (Avalanche photo diode) instead of photomultiplier tubes (PMTs) for fluorescence detection.  This is a revolutionary concept reduces the physical size and complexity of the instrument without compromising sensitivity and quality.  This webinar will include animated videos to help explain this new approach to cytometry.  At the end you will understand how new technology has been incorporated into the CytoFLEX flow cytometer to give you the edge in your research.

Learning Objectives:

• Learn about an innovative approach to a traditional technology which encompasses several advanced features that are unusual for a flow cytometer.  For example, the CytoFLEX includes a communications technology called Wavelength Division Multiplier (WDM) that changes the paradigm of light collection techniques in flow cytometry
• Learn about a revolutionary concept that reduces the physical size and complexity of the instrument without compromising sensitivity and quality
• Understand how new technology has been incorporated into the CytoFLEX flow cytometer to give you the edge in your research 

We compared the workflow of the Beckman Coulter automated Aquios CL flow cytometer, using AQUIOS Tetra software, to a single platform method using the Beckman Coulter TQ-Prep lysing system with subsequent analysis on the Beckman Coulter FC500 flow cytometer with Tetra CXP software. 

Clinical patient samples were run on both the Aquios CL and the FC500 flow cytometers in a multi scenario setting to compare turn around time, time to first result and total elapsed time including the amount of hands-on time and procedural steps required to perform key tasks such as instrument start-up, compensation, quality control, specimen processing and instrument shutdown. 

The study results showed a great advantage to using the Aquios CL for automated lymphocyte subset enumeration. Across the board, the Aquios CL flow cytometer has shown increased efficiency with a decreased amount of hands-on time and procedural steps required to analyze specimens.  The ease of use, speed and knowledge required to run the Aquios CL flow cytometer make this instrument a good choice for both busy and remote clinical laboratory locations where experienced flow cytometry technologists are not readily available.

Learning Objectives:

• See a comparison of the workflow of the Beckman Coulter automated Aquios CL flow cytometer, using AQUIOS Tetra software, to a single platform method using the Beckman Coulter TQ-Prep lysing system with subsequent analysis on the Beckman Coulter FC500 flow cytometer with Tetra CXP software
• See turnaround time, time to first result and total elapsed time including the amount of hands-on time and procedural steps required to perform key tasks such as instrument start-up, compensation, quality control, specimen processing and instrument shutdown Clinical patient samples were run on both the Aquios CL and the FC500 flow cytometers in a multi scenario setting to compare
• See advantages to using the Aquios CL for automated lymphocyte subset enumeration

November 5, 2015, 8:00am PT, 11:00am ET, 3:00pm GMT

T-cells form an integral part of the human immune system by fighting off infection and eliminating transformed cells. Over the last decades numerous T-cells subsets with different functions and abilities have been identified (Farber et al. Nat Rev Immunol 2014). The wide spread use of monoclonal antibodies and multi-color flow cytometry allows for the routine identification of these subsets based on their immune-phenotype (Chattopadhyay et al. Cytometry 2012). However, the increasingly detailed understanding and the dynamic nature of different T-cell subsets also results in a dramatic increase in the complexity of the antibody panels used.

Here, we will demonstrate the identification of naïve, memory and effector T cells at various stages of differentiation, but also the detection of rare populations such as stem cell like memory T cells and different Treg subpopulations using a 13-color marker combination that allows for the identification of T cell subtypes according to classical and more recent characterization criteria. We will outline the panel design rational taking into account expression patterns as well as fluorochrome characteristics. The applied gating strategies will be discussed.

Learning Objectives:

• Demonstrate the identification of naïve, memory and effector T cells at various stages of differentiation, but also the detection of rare populations such as stem cell like memory T cells
• Understand the panel design rationale, taking into account expression patterns as well as fluorochrome characteristics 

A fundamental aspect of the thermodynamic characterization of macromolecular complexes is the determination of the component stoichiometry. There are a number of approaches that may be employed in this effort, including mass spectrometry, light scattering, and sedimentation equilibrium analytical ultracentrifugation (AUC). In recent years, an AUC method called multi-signal sedimentation velocity (MSSV) has emerged as an excellent means to elucidate stoichiometry. This technique, pioneered in the laboratory of Peter Schuck, capitalizes on the Beckman XL-A/I centrifuge’s ability to acquire sedimentation velocity (SV) data using multiple wavelengths and, in the case of the XL-I, multiple optical systems. Using the popular c(s) formalism, MSSV can supply the molar ratio of 2 – 4 species in a stable, co-sedimenting complex. In conjunction with the hydrodynamic information inherently available from a SV experiment, this often allows the unambiguous determination of stoichiometry. Basic theory and experimental design for MSSV will be featured in this webinar, along with the analytical strategies necessary to produce reliable results. Also, advanced topics, including measures that may be taken in difficult cases, will be covered. Throughout, concrete, real-world examples of MSSV will be presented.

Learning Objectives:

• Learn why an analytical ultracentrifugation (AUC) method called multi-signal sedimentation velocity (MSSV) has emerged as an excellent means to elucidate stoichiometry in recent years 
• Learn the basic theory and experimental design for MSSV, along with the analytical strategies necessary to produce reliable results.
• See real-world examples of MSSV application

During the development of oral cancer (OC), cancer-associated fibroblasts (CAFs) create a supporting niche by maintaining a bidirectional crosstalk with cancer cells, mediated by classically secreted factors and nanometer-sized vesicles, called exosomes. Little is known about the molecular alterations in CAFs that determine their unique functional properties and how exosomes contribution to OC progression. To investigate stromal heterogeneity in OCs we isolated and characterized human fibroblasts from resected tumors (CAFs) and adjacent tissue (AFs). We employed shotgun proteomics to identify CAF-associated proteins in total lysate, conditioned media and exosomes. Our comprehensive dataset of 4247 proteins represents a detailed signature of a pro-tumorigenic stroma. Gene ontology enrichment analysis has shown that proteins involved in regulation of cancer cell migration are enriched in the exosomal fraction. To validate our findings we have shown a significant effect of CAF-derived exosomes on OC cells motility, proliferation and the activation of the MAPK pathway. Quantitative proteomics was then applied to highlight differentially expressed proteins in exosomes from CAFs and AFs and generate a short list of proteins specific to the CAF-like state. Significant differences were observed in proteins involved in metabolism, suggesting that metabolic synergy between CAFs and cancer cells could involve exosomal cargo. In summary, integrative proteomics identified a signature of CAF-enriched exosomal proteins involved in pathways mediating tumor-stromal crosstalk. Here, we show that fibroblast-secreted exosomes affect OC cells growth, motility and glucose metabolism. Our data provide a foundation to investigate how the tumor microenvironment affects OC progression.

Learning Objectives:

• Learn how quantitative proteomics is applied to highlight differentially expressed proteins in exosomes from cancer-associated fibroblasts (CAFs) and adjacent tissue (AFs)
• Learn how fibroblast-secreted exosomes affect OC cells growth, motility and glucose metabolism

October 7, 2015, 8:00am PT, 11:00am ET, 3:00pm GMT

There are many laboratory Total Organic Carbon (TOC) analyzers available, most of which appear to be suitable for pharmaceutical QC use, at least on paper! However many turn out to be unsuitable, either just too difficult to validate to pharmaceutical requirements reliably, or because they do an indirect measurement and calculate TOC and hence frequently report negative TOC values. This webinar guides you through the different TOC analyzer measurement technologies and explains why certain technologies are best suited to pharmaceutical applications and shows how the new QbD1200 TOC analyzer from Beckman Coulter makes a direct TOC measurement to avoid many of the pitfalls of this challenging application.

Learning Objectives:

• Understand the different TOC analyzer measurement technologies
• Understand why certain technologies are best suited to pharmaceutical applications

Background:  The extracellular vesicle (EV) research field has dramatically increased in the last five years.  Using a high-speed flow cytometric sorter, EVs may be isolated at high rates such that researchers can differentially separate, isolate and characterize the EVs for downstream analysis.  EVs contaminated with proteins, dye or antibody aggregates of the same size, but different mass, can be characterized based on these physical properties in the analytical ultra-centrifuge. This presentation focuses on characterizing the EVs on an XLA/I AUC and dynamic light scatter isolated by flow cytometric sorting. Methods:  Extracellular vesicles were isolated from a HeLa cell line using serial ultracentrifugation.  HeLa EVs were measured for total protein content and confirmed using CD63 Dyna-beads.  EVs were stained with PKH26 and sorted on the MoFlo Astrios EQ using side scatter and fluorescence.  Instrument performance was tested with polystyrene latex beads ranging from 22- 104 nm, and 100 nm liposomes for minimum detection limits.  Absorbance and interference were used on AUC to measure the EV sedimentation.  DelsaMax (DM) Core, dynamic light scatter (DLS) instruments, were used to measure the particle sizes of EV samples.  Results:  The Astrios EQ was able to distinguish and sort the EVs with some overlap on SSC noise.  100 nm liposomes and 81 nm were visible above noise.  EVs stained with PKH-26 and sorted on SSC and fluorescence with populations above and in the noise.  Sorted fractions were analyzed on the AUC and DM for unstained, stained and aggregate population distributions.  AUC indicated EV populations with varied sizes and dye aggregates and EV size and distribution was measured by the DM with DLS.   Conclusions:  Sorting stained EV populations on a high speed sorter provides for their visualization, separation and characterization.  The AUC effectively separated particles on their sedimentation velocity and clarified issues with dye aggregation vs EV staining.  Addditionally, the DelsaMax allowed for quick analysis of post-sorted populations. The Astrios EQ was able to sort EVs and AUC provided additional analysis for exosome purity.

Learning Objectives:

• Learn how EVs may be isolated at high rates such that researchers can differentially separate, isolate and characterize the EVs for downstream analysis using a high-speed flow cytometric sorter
• Learn how EVs contaminated with proteins, dye or antibody aggregates of the same size, but different mass, can be characterized based on these physical properties using the analytical ultracentrifuge
• This presentation focuses on how to characterize EVs using an XLA/I AUC and dynamic light scatter isolated by flow cytometric sorting

 Beckman Coulter, the stylized logo, Astrios, and DelsaMax are trademarks of Beckman Coulter, Inc.  Beckman Coulter and the stylized logo are registered in the USPTO.

Micro RNAs (miRNAs) are small, naturally-occurring non-coding ribonucleic acids with sizes between 18 and 40 nucleotides (nts) that have been demonstrated to play a significant role in the regulation of gene expression.  As a result, interest in smaller RNA species such as miRNA has increased. This webinar describes the method development for miRNA purification, enrichment and RNA fractionation using Beckman Coulter’s SPRI (Solid Phase Reverse Immobilization) magnetic bead based chemistry and the Biomek automated extraction method.  Total RNA and miRNA can be purified from very small amounts of highly degraded FFPE (Formalin Fixed, Paraffin Embedded) and LCM (Laser Captured Microdissection) samples. The SPRI chemistry produced superior results to a column purification method for miRNA purification, miRNA enrichment and RNA size selection.  The SPRI chemistry is an easy, high-yielding and automation-friendly nucleic acid purification process that does not require centrifugation and vacuum filtration steps. Purified nucleic acids are easily eluted from the magnetic beads under aqueous conditions, which provide maximum flexibility for downstream applications.

Learning Objectives:

• Understand the method development for miRNA purification, enrichment and RNA fractionation using Beckman Coulter’s SPRI (Solid Phase Reverse Immobilization) magnetic bead based chemistry and the Biomek automated extraction method
• Learn how total RNA and miRNA can be purified from very small amounts of highly degraded FFPE (Formalin Fixed, Paraffin Embedded) and LCM (Laser Captured Microdissection) samples

The webinar will start at 4am Pacific Time, 7am Eastern Time, 1pm Central European Summer Time, 7pm China Standard Time

 

Extracellular vesicles (EVs) including exosome and microvesicles are nanometer sized vesicles released by cells as a novel form of intercellular communication without direct cell-to-cell contacts, delivering lipids, cellular proteins, mRNAs and non-coding RNAs. Recent studies have shown that EV plays an important role in diseases, as well as been employed as a vehicle for targeted delivery of therapeutic genes/drugs. Accurate spatiotemporal assessment of nanosized EV delivery and cargo RNA translation, however, requires specific and robust imaging technologies. For fluorescent reporters conjugated to proteins enriched in EVs, labeling may be restricted to subpopulations of EVs, limiting their wider use to observe multiple EV types. Meanwhile, fluorescent dyes for EV lipid-labeling including PKH may not reflect true half-life of EVs and can be retained in association with other lipid entities for long periods, thus misguiding spatiotemporal assessment of EV dynamics, especially over extended periods. This webinar will discuss emerging methods developed for EV visualization, as well as findings made from employing these new technologies.

Learning Objectives:

• Discuss emerging methods developed for EV visualization
• Understand findings made from employing new technologies developed for extracellular vesicle (EV) visualization

Analytical ultracentrifugation (AUC) is a powerful technique for analyzing fundamental characteristics of macromolecules in solution, including size and shape, assembly state, and degree of polydispersity. It allows the study of conformational changes and complex formation in physiologically relevant buffers. AUC is useful as a method to characterize structural aspects of macromolecules and as a quantitative technique that provides access to detailed thermodynamic characterization of a system under study. This webinar will describe basic principles underlying sedimentation velocity and sedimentation equilibrium AUC methods and ways in which AUC can be used to complement crystallographic or other structural biology approaches. Examples will be provided to illustrate AUC approaches for characterization of monodisperse or assembling proteins, simultaneous size-and-shape analysis of multi-species mixtures, and determination of affinity and stoichiometry of macromolecular complexes.

Learning Objectives:

• Learn about basic principles underlying sedimentation velocity and sedimentation equilibrium AUC methods and ways in which AUC can be used to complement crystallographic or other structural biology approaches
• Learn AUC approaches for characterization of monodisperse or assembling proteins, simultaneous size-and-shape analysis of multi-species mixtures, and determination of affinity and stoichiometry of macromolecular complexes

Exosomes are small secreted vesicles that have a diameter of ~50-200 nm. Exosomes are enriched for a specific subset of host-derived proteins, nucleic acids, lipids and carbohydrates, though they also incorporate most host cell molecules at baseline levels. Various models of exosome biogenesis have been proposed, but the field lacks the robust mechanistic studies that are needed to obtain a molecular understanding of vesicle secretion. To shed light on this process, our laboratory has taken a cargo-based approach in which we focus on the cis-acting signals that are necessary and sufficient for the budding of specific proteins. These studies have revealed that exosomal proteins are targeted to sites of vesicle budding by a combination of (1) high-order oligomerization and (2) binding to the plasma membrane. In addition, our work supports the hypothesis that the plasma membrane is a major site of exosome budding. Interestingly, HIV and other retroviruses have the same topology, size, and array of host cell molecules as exosomes, raising the possibility that retroviruses bud from infected cells by an exosomal pathway. This hypothesis is supported by the fact that retroviral Gag proteins, their main structural protein, are targeted to sites of exosome budding, bud from cells in association with exosomal cargo proteins, form high-order oligomeric complexes that bind the plasma membrane, and require plasma membrane binding in order to bud from cells. Modeling HIV budding as an exosomal process has led to new lines of experimentation, new sets of data, and new interpretations of decades-old observations, all of which provide increasing support for the hypothesis that HIV and other retroviruses exploit the exosomal machinery to generate infectious viral particles.

Learning Objectives:

• Various models of exosome biogenesis have been proposed, but the field lacks the robust mechanistic studies that are needed to obtain a molecular understanding of vesicle secretion. Learn how our laboratory has taken a cargo-based approach in which we focus on the cis-acting signals that are necessary and sufficient for the budding of specific proteins.
• Learn why our work supports the hypothesis that the plasma membrane is a major site of exosome budding

The emerging field of RNA nanotechnology has led to the utilization of the platform in the field of bionanotechnology as sensing platforms, diverse nanoparticle construction, in vivo computing, and even in vivo drug delivery. Purification of large quantities of supramolecular RNA complexes is of paramount importance due to the large quantities of RNA needed and the purity requirements for in vitro and in vivo assays. Purification is generally carried out by liquid chromatography (HPLC), polyacrylamide gel electrophoresis (PAGE), or agarose gel electrophoresis (AGE); however, difficulties exist with each technique. Here, we describe an efficient method for the large-scale purification of RNA prepared by in vitro transcription using T7 RNA polymerase by cesium chloride (CsCl) equilibrium density gradient ultracentrifugation and the large-scale purification of RNA nanoparticles by sucrose gradient rate-zonal ultracentrifugation or cushioned sucrose gradient rate-zonal ultracentrifugation. The isolation technique has broad-reaching applicability as production of large quantities of RNA are critical to functional assays involving RNA splicing, RNA secondary structure, anti-sense RNA, and RNA:protein interaction studies.

Learning Objectives:

• Learn about an efficient method for the large-scale purification of RNA prepared by in vitro transcription using T7 RNA polymerase by cesium chloride (CsCl) equilibrium density gradient ultracentrifugation and the large-scale purification of RNA nanoparticles by sucrose gradient rate-zonal ultracentrifugation or cushioned sucrose gradient rate-zonal ultracentrifugation
• Learn how this isolation technique has broad-reaching applicability as production of large quantities of RNA are critical to functional assays involving RNA splicing, RNA secondary structure, anti-sense RNA, and RNA:protein interaction studies

With the development of the XLA/XLI analytical ultracentrifuges and their exquisitely sensitive optics that can accurately detect absorbance signals in the range of 0.005 A to 1.5 A. and differences in Rayleigh interference patterns to at least 5 ug/ml of protein solute in solution, new methods for sedimentation data analysis have been developed making it possible to quantitatively determine the stoichiometry of reactions and their energies of interaction as well as solute masses. In this talk, we will consider some of the experimental approaches that Dr. John Burgner and others have used to characterize these processes over the past few years.

Learning Objectives:

• Learn new methods for sedimentation data analysis that have been developed making it possible to quantitatively determine the stoichiometry of reactions and their energies of interaction as well as solute masses
• Learn some of the experimental approaches used to characterize stoichiometry of reactions over the past few years

Extracellular vesicles (EVs) include the exosomes (30-100 nm) that are produced through the endocytic pathway via the multivesicular bodies and the ectosomes (100-1000 nm) that are released through the budding of the plasma membrane. Despite the differences in the mode of biogenesis and size, reliable markers that can distinguish between exosomes and ectosomes are non-existent. Moreover, the precise functional differences between exosomes and ectosomes remains poorly characterised. Here, using label-free quantitative proteomics, we highlight proteins that could be exploited as markers to discriminate between exosomes and ectosomes. For the first time, a global proteogenomics analysis unveiled the secretion of mutant proteins that are implicated in cancer progression through EVs. Follow up integrated bioinformatics and experimental approach highlighted that exosomes are more oncogenic than ectosomes. These findings ascertain that cancer cells facilitate oncogenesis by the secretion of mutant and oncoproteins into the tumor microenvironment via exosomes and ectosomes.

Learning Objectives:

• Learn how follow up integrated bioinformatics and experimental approach highlighted that exosomes are more oncogenic than ectosomes
• Learn how using label-free quantitative proteomics, proteins were highlighted that could be exploited as markers to discriminate between exosomes and ectosomes
• Learn how cancer cells facilitate oncogenesis by the secretion of mutant and oncoproteins into the tumor microenvironment via exosomes and ectosomes

Sea slugs (Mollusca, Gastropoda, Nudipleura) provide unique opportunities for studying the evolution of behavior at the levels of single neurons, neural circuits, and their synaptic connections. Of particular interest to us are the mechanisms by which analogous forms of swimming behavior, such as dorsal-ventral (DV) or left-right (LR) body flexions, evolved independently in certain sea slug clades. Transcriptome sequencing has been useful for this research in two ways: first, it provides a means for characterizing the phylogenetic relationships amongst sea slug species, where robust phylogenetic trees can be constructed using hundreds of orthologous gene sequences. Indeed, a strong understanding of species phylogeny is a necessary pre-requisite for formulating hypotheses about how DV and LR swimming might have evolved in different lineages. Secondly, transcriptome sequencing provides unprecedented access to gene sequences from the sea slug brain, which can be used to probe the molecular mechanisms by which single neurons are recruited into rhythmic circuits to drive analogous forms of swimming. To illustrate, the lab previously found that in two species that independently evolved DV swimming, homologous single neurons called C2 independently acquired sensitivity to serotonin (5HT) to potentiate their synapses and activate their corresponding DV swimming neural circuits. We are now testing the hypothesis that convergent alterations in 5HT receptor function/expression in C2 neurons is responsible for this synaptic potentiation, having identified from the transcriptomes full length sequences for all known molluscan 5HT receptors.

Learning Objectives:

• Learn how transcriptome sequencing has been useful for the research of sea slugs and the mechanisms by which analogous forms of swimming behavior, such as dorsal-ventral (DV) or left-right (LR) body flexions, evolved independently in certain sea slug clades
• Learn how transcriptome sequencing provides a means for characterizing the phylogenetic relationships amongst sea slug species, where robust phylogenetic trees can be constructed using hundreds of orthologous gene sequences.
• Learn how transcriptome sequencing provides unprecedented access to gene sequences from the sea slug brain, which can be used to probe the molecular mechanisms by which single neurons are recruited into rhythmic circuits to drive analogous forms of swimming

Centrifugal elutriation is a mature technology that has been used for purification of cell subsets such as monocytes based upon cell density and size, without the need for labeling. The technique has been widely used for synchronization of cells or to purify cells in distinct cell cycle phases. The specialized elutriation rotor allows loading material into the spinning elutriator chamber, and by varying the fluid flow rate introduced into the chamber to counter the centrifugal force, fractions of particles with predictable size can be removed.

This webinar will review the basic technology, and then illustrate various established applications of centrifugal elutriation. Previously reported applications, as well as new applications under development in our lab, are a logical adjunct to the cell sorting techniques offered in a flow cytometry laboratory.

Learning Objectives:

• Review the basic centrifugal elutriation technology
• Illustrate various established applications of centrifugal elutriation, including reported applications, as well as new applications under development in our lab

In recent years, sophisticated new methods for sedimentation data analysis have opened up new avenues for leveraging AUC technology in a wide range of technology sectors, including life science research, pharmaceutical QC, material, polymer and nano science. In this talk, Borries Demeler will highlight some of the approaches used in his lab to extract ever more information from sedimentation velocity experiments. In this talk he will address ways of accurately characterizing systems at high resolution that are heterogeneous in both size and shape, and present methods for measuring the strength of macromolecular interactions by fitting discrete binding models to whole boundary sedimentation velocity experiments.

Learning Objectives:

• Learn ways to accurately characterize systems at high resolution that are heterogeneous in both size and shape
• Learn approaches used to extract ever more information from sedimentation velocity experiments
• See methods for measuring the strength of macromolecular interactions by fitting discrete binding models to whole boundary sedimentation velocity experiments

This educational session will focus on some of the new demands in counting particles in liquids, with a focus on the challenges of preparing and sampling protein-based materials. USP has recently posted a new test for protein-based materials, known as USP <787>, and indicated that a companion informational chapter, USP <1787>, will be crafted to further describe methods and options for evaluating protein-based materials. This presentation will delineate the differences between the more well-known USP 788 and the new USP 789, focusing on critical sample preparation and handling to provide good data on particulate concentration in protein solutions. Two types of instruments that could be employed for this method are discussed and compared, one based on light obscuration and a second on the Coulter principle.

Learning Objectives:

• Learn about the new demands in counting particles in liquids, with a focus on the challenges of preparing and sampling protein-based materials
• Learn about the new test USP has recently posted for protein-based materials, known as USP <787>
• This presentation will delineate the differences between the more well-known USP 788 and the new USP 789, focusing on critical sample preparation and handling to provide good data on particulate concentration in protein solutions
• Learn about and compare two types of instruments that could be employed for this method, one based on light obscuration and a second on the Coulter principle

Every cell in the body can release several types of extracellular vesicles (EVs), often called ”exosomes” or ”microvesicles”. These vesicles carry a wide array of molecules, including functional proteins and enzymes, as well as different RNA species (mRNA, microRNA and other species), all protected by a membrane lipid bilayer. EVs have the capacity to deliver molecular cargo to recipient cells by multiple pathways, including fusion at the level of the cell membrane, or by being internalized by cells. Current research is suggesting a wide diversity of extracellular vesicles , with diverse morphology according to electron microscopy studies, and difference in RNA and protein cargo according to bioinformatics analyses.

EVs have been implicated in several ways in disease. For example, in cancer, it has been shown that the number of EVs increase in the circulation. EVs can propagate disease in several ways, for example in cancer by enhancing angiogenesis and propagating metastasis, as well inducing immune tolerance. Furthermore, several studies today have suggested that the cargo of EVs released in disease states such as cancer, including proteins and RNA, will fundamentally change, which can explain changes in function.

This lecture will lay the background to EVs and will discuss extensively the diversity of EVs released by single cell types in culture, and diversity of EVs in body fluids, discussing their relevance for cell-to-cell communication.

Learning Objectives:

• Learn about the study of extracellular vesicles and their relevance for cell-to-cell communication
• Learn about how extracellular vesicles have been implicated in several ways in disease
• Understand the wide diversity of extracellular vesicles, with diverse morphology according to electron microscopy studies, and difference in RNA and protein cargo according to bioinformatics analyses

Advances in stem cell biology have raised great expectations that diseases of the central nervous system may be ameliorated by the development of non-haematopoietic stem cell medicines. Yet, the application of stem cells as therapeutics is challenging and the interpretation of some of the outcomes ambiguous. The initial idea that stem cell transplants work only via structural cell replacement has been challenged by the observation of consistent intercellular information exchange between the graft and the host. Sustained stem cell graft-to-host exchange of signals has led to remarkable trophic effects on endogenous brain cells and beneficial modulatory actions on innate and adaptive immune responses that ultimately promote the healing of the injured CNS. Among a number of promising candidate stem cell sources, mesenchymal/stromal stem cells (MSCs) and neural stem/precursor cells (NPCs) are being extensively investigated for their capacities to signal to the immune system upon transplantation in experimental CNS diseases.
 

Here, we focused on defining whether the form of cellular signalling mediated by extracellular membrane vesicles (EVs) exists for neural stem/precursor cells (NPCs), and on its molecular signature and functional relevance on target cells. We also investigated whether the EV cargo molecules are modulated by extracellular pro- or anti-inflammatory cytokines, determined the key elements responsible for this novel mechanism of EV-mediated intercellular communication, and finally reflected on the forthcoming challenges related to the translation of these exciting experimental proofs into ready-to-use clinical medicines for inflammatory CNS diseases.

Learning Objectives:

• Learn about whether the EV cargo molecules are modulated by extracellular pro- or anti-inflammatory cytokines, and the key elements responsible for this novel mechanism of EV-mediated intercellular communication
• Define whether the form of cellular signaling mediated by extracellular membrane vesicles (EVs) exists for neural stem/precursor cells (NPCs), and on its molecular signature and functional relevance on target cell
• Understand challenges related to the translation of these exciting experimental proofs into ready-to-use clinical medicines for inflammatory CNS diseases

 

In this webinar, the presenter will discuss:

 

   An Introduction to EPFL’s Core Facility and its role in furthering flow cytometry research

   Workflow options for flow cytometry and multicolor assays

   Applications for Gallios combined with Kaluza Analysis and Kaluza Acquisition Software

   Benefits for core facilities, labs and companies

   Audience Q&A

 

This webinar is ideal for

   Flow Cytometry researchers of all levels

   Core Laboratory Managers

   Flow cytometry core facility staff

Learning Objectives:

• Understand workflow options for flow cytometry and multicolor assays
• Learn about applications for Gallios combined with Kaluza Analysis and Kaluza Acquisition Software
• Learn about benefits of using Gallios combined with Kaluza Analysis and Kaluza Acquisition Software for core facilities, labs and companies

 

Successfully performing high throughput DNA purification in a clinical laboratory requires a robust automated method to handle the wide ranges in DNA quality and quantity found with patient buccal swabs, and to avoid the difficulties of sample recollection. Adopting a single, scalable method ensures stable workflow with consistent patient report turnaround time, even during spikes in incoming sample numbers, and greatly simplifies the CLIA regulated validation process. A single Biomek FX workstation configured with Beckman Coulter’s DNAdvance magnetic bead method enables the purification of approximately 2000 samples during an 8 hour shift with a combined 99.8% success rate per patient, while protecting laboratory personnel from infectious agent exposure and repetitive stress injuries. Special considerations and solutions for clinical environments will be presented that also apply to high throughput research laboratories. 

Learning Objectives:

• Learn how a single Biomek FX workstation configured with Beckman Coulter’s DNAdvance magnetic bead method enables the purification of approximately 2000 samples during an 8 hour shift with a combined 99.8% success rate per patient, while protecting laboratory personnel from infectious agent exposure and repetitive stress injuries
• Learn about special considerations and solutions for clinical environments that also apply to high throughput research laboratories

 

  Sedimentation velocity (SV) experiments on dispersed nanoparticles are a powerful method for determining otherwise hard to evaluate properties of the nanoparticle such as the density and size of the adsorbed interfacial layer, or for a direct evaluation of the entire distribution of a property such as particle size. The ability to measure these properties in situ in either aqueous or non-aqueous media, and via multiple detection techniques, provides information not readily available through other methods such as electron microscopy. My research has focused in particular on utilizing the capabilities of sedimentation velocity methods for the characterization of single-wall carbon nanotubes (SWCNTs).

  Single-wall carbon nanotubes are a family of chemical species comprised entirely of a “rolled up” single layer hexagonal lattice of carbon. Depending on the diameter of the formed cylinder, and the vector of the hexagonal lattice relative to the tube axis, these nanoparticles can be metallic, semi-metallic or semi-conducting, each having unique optical, mechanical, thermal, and electrical properties. Many of the applications for this set of materials however depend on the properties of the material when dispersed in the liquid phase, or contain liquid phase processing steps. The key aspects of liquid phase dispersion and processing though hinge on controlling the properties of the interfacial layer between the nanotube and the solution to enable dispersion, selective fractionation and/or biocompatibility. Density contrast variation methods combined with sedimentation velocity experiments provide a high resolution methodology for evaluating this interface. I will present results detailing the use of such density contrast techniques on the characterization of the interfacial layer on specific SWCNT species that demonstrate the power of analytical ultracentrifugation to resolve this interfacial layer.

  Separately, a second critical factor for SWCNT use in dispersion form is the size distribution of the dispersed population; this distribution strongly affects the transport of the nanotubes in many environments as well as the achievable properties in end applications, and is thus a necessary parameter for quality control. Measuring the whole size distribution of a nanoparticle is however typically very challenging for non-monodisperse particles. In a second set of results, I will show that the distribution of sedimentation coefficients for SWCNTs can be used to calculate through hydrodynamic theory the size distribution of the population in the dispersion in quantitative agreement with other techniques such as atomic force microscopy (AFM).
What you will learn:

  Methodology for the characterization of an important class of nanomaterials via SV, particularly involving variations of density contrast experiments to elucidate different interfacial parameters.
Discussion of concerns, details, assumptions, and problems in utilizing SV for nanoparticles, including some considerations for overcoming these issues through experimental methodology.

Learning Objectives:

• Understand how sedimentation velocity (SV) experiments on dispersed nanoparticles are a powerful method for determining otherwise hard to evaluate properties of the nanoparticle such as the density and size of the adsorbed interfacial layer
• Understand how to utilize the capabilities of sedimentation velocity methods for the characterization of single-wall carbon nanotubes (SWCNTs)

 

AUC sedimentation velocity (SV) has a longstanding reputation as being the gold standard for the matrix free quantification of aggregates in biopharmaceuticals. The continuous c(S) distribution analysis as implemented in the software SEDFIT is the most commonly used data evaluation method in the biopharma environment due to regulatory requirements and the reliable reporting of very minor species by the algorithm. I will show how to set up such an analysis in order to achieve enhanced reproducibility during a study of many batches of one protein. Attention will be also paid to the recently developed calibration procedures regarding the temperature, scan time and the radial calibration of the centrifuge.
In the second part examples are shown apart from the generic method of quantitating aggregates in dilute solutions under standardized conditions. These examples will include the determination of the extinction coefficient and concentration of protein formulations, a task far from being trivial in the biopharmaceutical field and the determination of the second virial coefficient, which is known as a predictor for aggregation propensity. The presentation will conclude with some applications of sedimentation equilibrium (SE) for the screening of weak interactions and the determination of detergent binding to proteins.

Learning Objectives:

• Learn how to set up continuous c(S) distribution analysis as implemented in the software SEDFIT in order to achieve enhanced reproducibility during a study of many batches of one protein
• See some applications of sedimentation equilibrium (SE) for the screening of weak interactions and the determination of detergent binding to proteins

 

Colloidal nanoparticles have become important materials in biomedicine, catalysis, and optoelectronics. Their unique nanoscale properties stem from their size (particularly of the inorganic particle core) and surface composition of their organic ligand-shell. Given the polydispersity of nanoparticles, and complex composition, no technique other than Analytical Ultracentrifugation (AUC) has the potential to provide detailed information on the overall particle size, density, molecular weight and aggregation state in solution.

While AUC has become a standard tool for bioscientists to quantitatively study the size, confirmation, and interaction of macromolecules and proteins in solution for many decades, it has not had the same success with nanoparticles. Unlike nanoparticles, most proteins have a well-known partial specific volume, and hence it’s straightforward to convert quantities measured from AUC, such as the sedimentation coefficients, into parameters that provide physical insights into the macromolecule, such as frictional ratio (i.e. shape) and molecular weight.

Recent advancements in the mathematical modeling of the sedimentation boundaries have enabled the parameterization of the sedimentation and diffusion coefficients of all the species detected in solution during centrifugation in terms of their partial specific volume and molecular weight. This advancement opens the door to quantitative investigation of most nanoparticle systems by AUC - without a priori knowledge of their partial specific volume - since most nanoparticles have a constant shape which can be assessed by electron microscopy.

In this tutorial webinar I will introduce some of the latest methods and procedures to analyze nanoparticles in solution with AUC. I will also provide insights on avoiding common experimental challenges that researchers face when studying nanoparticles with the AUC, which may lead to artifacts in the experimental data.

Learning Objectives:

• Learn about the latest methods and procedures to analyze nanoparticles in solution with Analytical Ultracentrifugation (AUC)
• Learn insights on avoiding common experimental challenges that researchers face when studying nanoparticles with the AUC

The therapeutic utility of stem cells is rooted in an understanding -- and exploitation -- of their natural role from earliest development to lifes end. Their job is first to participate in organogenesis and then to maintain homeostasis of that organ (e.g., the nervous system) in the face of perturbations. Accomplishment of these goals requires numerous actions, cell replacement representing but one. The tasks, in fact, require extensive cross-talk between multiple cell types (including stem cell-derived progeny themselves) and the unfolding of complex developmental  programs. This complexity actually enriches the therapeutic potential of the stem cell. 
We study the behavior of neural stem cells (NSCs) in various models of injury and degeneration. During neurodegeneration and inflammation, factors are transiently elaborated which draw NSCs (even over great distances) to engage the niche and attempt restoration of equipoise by a variety of mechanisms. These actions include differentiating towards the replacement of impaired neural cells, both neurons and non-neuronal chaperone cells, all of which are essential for restitution of function. NSCs elaborate factors that promote neuroprotection, trophic support, differentiation, neuritogenesis, connectivity, angiogenesis, inhibition of inflammation and scarring. In addition to producing diffusible factors, NSCs communicate via gap junctions to re-equilibrate the intracellular metabolism of endangered neurons. NSCs may serve as vehicles for protein delivery enabling simultaneous cell and gene therapy. NSCs synergize with biomaterials to "re-engineer" damaged regions. Multimodal approaches are likely required for most neurological conditions; NSCs may serve as the glue. When studied in vitro (development- or disease-in-a-dish), NSCs may help identify novel mechanisms, drug targets, and the drugs themselves.

Learning Objectives:

• Understand the therapeutic utility of stem cells
• Learn about the behavior of neural stem cells (NSCs) in various models of injury and degeneration

Abstract:
•General background, diagnostic and bio-marker implications of exosomes research
•Current solutions to purifying and identifying exosomes rapidly and accurately
•Recent applications work for improving exosome isolation using automated liquid handling and ultracentratigation, to diminish human variability in density gradient preparation, and improve overall research workflow
•Overview of ultra-low volume, highly sensitive dynamic light scattering as a reliable and rapid option for exosome size characterization

Learning Objectives:

• Learn about the general background, diagnostic and bio-marker implications of exosomes research
• Learn about current solutions to purifying and identifying exosomes rapidly and accurately
• Learn about recent applications work for improving exosome isolation using automated liquid handling and ultracentratigation, to diminish human variability in density gradient preparation, and improve overall research workflow
• See an overview of ultra-low volume, highly sensitive dynamic light scattering as a reliable and rapid option for exosome size characterization

Dr. Johannes van der Loo will cover the vital role viral vectors play in today's gene research, as well as the use of ultracentrifugation to produce these key delivery systems.

Learning Objectives:

• Understand the vital role viral vectors play in today's gene research
• Understand the use of ultracentrifugation to produce these key delivery systems

Abstract:

• Understand unique attributes of diamond-based nanoparticles and why it is used for imaging for cancer and drug delivery
• Understand in-vitro and in-vivo applications of diamond-based nanoparticles

Learning Objectives:

• Understand unique attributes of diamond-based nanoparticles and why it is used for imaging for cancer and drug delivery
• Understand in-vitro and in-vivo applications of diamond-based nanoparticles