Introduction to Air Monitoring

Over the years, extensive evaluations of columns manufactured with ionic liquid stationary phases have occurred. Their main strength was discovered to be unique selectivity. This selectivity is made possible due to the various combinations of cations and anions that are available along with spacer groups used to prepare these germinal
dicationic phases. Columns prepared with di- or tricationic phases have the ability to perform many of the same applications as columns made with polysiloxane polymer or polyethylene glycol stationary phases of similar polarity, but with slight elution order changes. Many times this results in increased resolution and/or shorter run times. This webinar will compare and contrast the selectivity of the ionic liquids stationary phases with
traditional phases of similar or like selectivity’s for applications with a
variety of different sample types from a number of industries including
petrochemical, pharmaceutical, environmental, food and beverage and flavor and fragrance.

This presentation will focus on the current trends in hepatic culture technologies and considerations for how they are impacted by the quality and performance of the cell materials used. The current state-of-the-art in procurement, production and characterization of primary hepatocytes for in vitro research applications will be reviewed, and measures for improving the validation and qualification of hepatic cells for specific applications also will be proposed.

Duolink® proximity ligation assay (PLA) technology allows you to visualize protein interactions with cellular localization and quantities by amplifying signals corresponding to single and post-translational protein events. With 1000x sensitivity and high specificity, this protein detection technology allows to visualize protein functions, all within a native cell. The PLA method provides:
- Visual protein interactions – both stable and transient
- Endogenous protein detection – no overexpression or genetic manipulation
- High specificity – use of two antibodies/probes eliminates false positives
- Single molecule sensitivity – rolling circle amplification makes proteins visible
- No special equipment needed – standard immunofluorescence methods

This webinar will review how to Work with PLA technology and provide an overview of their potential along with example applications.

Topics covered:
- How to work with Duolink PLA technology
- Overview of most relevant applications using Duolink
- Product offering to run an assay with PLA using Immunofluorescence or Flow Cytometry

Gene editing has become a firmly established technology within the discovery sciences arena. With the advent of CRISPR/Cas9 systems, the researcher's ability to find an active nuclease for nearly any region of any genome is now a reality. Even with better nucleases available, those who routinely use gene editing tools to manipulate cell lines encounter other significant challenges that pose a barrier to building the "correct" cell line model. As a well-established partner for custom cell line engineering, we at MilliporeSigma have encountered many of these challenges and have developed and/or implemented a number of methods to circumvent them. Several of these methods and the new tools available for gene editing will be discussed in this webinar, along with a summary of how they have impacted our internal cell line engineering programs.

Single molecule counting (SMC™) technology enables precise measurement of molecules at levels previously undetectable, down to the femtogram/mL levels, allowing researchers to identify new biomarkers, or assist in therapeutic development with an improved view of efficacy, safety & time course studies. When time and resources are limited, Merck KGaA offers a comprehensive portfolio of Custom Services supported by a scientific team with core expertise in SMC™ technology. Learn how our team will partner with you to develop a project specific to your requirements, whether that is fit-for-purpose sample testing, biomarker analysis using our current SMC™ immunoassays, or development and manufacture of an immunoassay for your novel target of interest. Learn how we work with our clients to define and tailor a customized project plan that includes milestone driven tasks, collaborative data review and progress reports. Whether your focus is to expedite your clinical research or to transfer a method to a CRO, we will show you how our services can help you accelerate programs from discovery into clinical trials.

Single molecule counting (SMC™) technology enables precise measurement of molecules at levels previously undetectable, down to the femtogram/mL levels, allowing researchers to identify new biomarkers, or assist in therapeutic development with an improved view of efficacy, safety & time course studies. When time and resources are limited, Merck KGaA offers a comprehensive portfolio of Custom Services supported by a scientific team with core expertise in SMC™ technology. Learn how our team will partner with you to develop a project specific to your requirements, whether that is fit-for-purpose sample testing, biomarker analysis using our current SMC™ immunoassays, or development and manufacture of an immunoassay for your novel target of interest. Learn how we work with our clients to define and tailor a customized project plan that includes milestone driven tasks, collaborative data review and progress reports. Whether your focus is to expedite your clinical research or to transfer a method to a CRO, we will show you how our services can help you accelerate programs from discovery into clinical trials.

Innate immune cells play a critical role in cell-mediated immunity and have the potential to serve as cell-based therapies to treat a broad spectrum of immune diseases such as cancer and autoimmune disorders. Modified immune cells, such as genetically engineered CAR-T cells, have proven to be critical in developing new cell-based therapies for these diseases. However, immune cell biology creates challenges during the gene-editing process that lead to hyper-regulated RNA and DNA sensing pathways and enhanced cell death upon introduction of exogenous ribonucleotides. Further, engineering in primary immune cells is often restricted due to their limited expansion capacity. Genetic engineering in immune cells has traditionally relied on random integration of gene-editing components using viral delivery systems. In contrast, genome editing mediated by nucleases, such as CRISPR/Cas9-single guide RNPs, provide a platform for precision editing, and alleviate the potential side effects caused by randomly integrated viral DNA. While RNP gene editing in immune cells is just beginning to be considered by the immune-therapeutics field, our recent advances demonstrate that this approach can be used to create targeted modifications in two key cell types, the macrophage and the CD8+ primary T-cell. In an effort to circumvent challenges with the finite lifespan of primary T-cells, we targeted genes to edit that rendered this cell type “pseudo-immortalized”, allowing additional passages for further downstream genome editing and propagation. In addition, we demonstrated that precision editing can be used to introduce disease relevant SNPs into the macrophage genome, which resist introduction of exogenous ribonucleotides due to the induction of apoptotic pathways. Advances such as these overcome many of the obstacles currently faced with immune cell editing and offer improved gene stability and expression in immune cells and will transform the Immuno-Oncology and Gene Therapy fields.

Innate immune cells play a critical role in cell-mediated immunity and have the potential to serve as cell-based therapies to treat a broad spectrum of immune diseases such as cancer and autoimmune disorders. Modified immune cells, such as genetically engineered CAR-T cells, have proven to be critical in developing new cell-based therapies for these diseases. However, immune cell biology creates challenges during the gene-editing process that lead to hyper-regulated RNA and DNA sensing pathways and enhanced cell death upon introduction of exogenous ribonucleotides. Further, engineering in primary immune cells is often restricted due to their limited expansion capacity. Genetic engineering in immune cells has traditionally relied on random integration of gene-editing components using viral delivery systems. In contrast, genome editing mediated by nucleases, such as CRISPR/Cas9-single guide RNPs, provide a platform for precision editing, and alleviate the potential side effects caused by randomly integrated viral DNA. While RNP gene editing in immune cells is just beginning to be considered by the immune-therapeutics field, our recent advances demonstrate that this approach can be used to create targeted modifications in two key cell types, the macrophage and the CD8+ primary T-cell. In an effort to circumvent challenges with the finite lifespan of primary T-cells, we targeted genes to edit that rendered this cell type “pseudo-immortalized”, allowing additional passages for further downstream genome editing and propagation. In addition, we demonstrated that precision editing can be used to introduce disease relevant SNPs into the macrophage genome, which resist introduction of exogenous ribonucleotides due to the induction of apoptotic pathways. Advances such as these overcome many of the obstacles currently faced with immune cell editing and offer improved gene stability and expression in immune cells and will transform the Immuno-Oncology and Gene Therapy fields.

Whether you are looking to knockout, knockdown, or overexpress genes, lentiviral transduction is the superior mechanism for delivering genetic cargo into hard to transfect cells and in vivo systems. Lentivirus is a perfect tool for screening applications since the delivered genetic material is constitutively expressed by the cells long-term. We will discuss the flexibility of our expert manufacturing group and present examples of applications suitable with lentivirus.

During this webinar, we dispel the preconceived misconception that lentivirus is risky or cumbersome to use. As a trusted lentiviral manufacturer, we will share our best practices for handling lentivirus and the simple steps that set you up for success.

According to the World Health Organization (WHO) cardiovascular diseases (CVD’s) are the leading cause of death accounting for more than 17.3M deaths globally. Modern cardiac diagnostics tests and monitoring techniques are providing ever increasing insight into the health of the human heart. In this presentation we examine some of the new and emerging cardiac biomarkers that could complement existing diagnostic and prognostic methods and have the potential to revolutionize our current understanding of cardiac health.

Leukemia and lymphoma are hematologic neoplasms that affect members of all age groups. Each year, over 140,000 people in the US are diagnosed with a hematologic malignancy of some kind. With constant advancement of treatment options, the importance of accurate diagnosis and detection of lymphomas and leukemias becomes more and more relevant to the survival of the patient, and immunohistochemistry has served as a key auxiliary test in determining these diagnoses. This presentation covers many of the basic science, facts, and statistics of hematologic malignancies, as well as the utility of immunohistochemical testing with markers such as CD20, PAX-5, CD61, CD71, Cyclin D1, and SOX-11 in the accurate diagnosis and survival rates of lymphoma and leukemia.

T cell biology is integral to the study of normal immune regulation as well as cancer biology, Car-T cells, epitope specificity and antigen presentation. However, primary T cells can be difficult to propagate in culture for the length of time necessary for functional assays. In addition, primary T cells express variant T cell receptor (TCR) heterodimers that can be challenging to identify and may not be optimal for downstream studies. We sought to simplify this system using transformed T cells which can be grown in culture for extended periods of time. We engineered a floxed landing pad sequence into the safe harbor AAVS1 genetic locus using CompoZr zinc finger nucleases. Both the promoter and landing pad expression cassette are flanked by unique lox sites, allowing swapping of either the promoter and/or expression cassette as needed. We ensured that only one copy of this sequence was found within the genome to avoid any complications associated with random insertion events. We also generated a landing pad cell line null for the endogenous TCR using Cas9/CRISPR ribonucleotide complexes. Both the TCR alpha and beta loci were rendered null due to non-homologous end joining and the presence of insertions and deletions culminating in premature stop codons were genotyped using next generation sequencing. The absence of a functional TCR was validated using flow cytometry staining for surface TCR and CD3. This cell line was then used to generate a knock-in of the desired exogenous TCR heterodimer to the landing pad locus, verified using flow cytometry staining. These lines will be very useful for a multitude of studies where a researcher needs to express a gene of interest in a discrete genetic locus or wants to generate a panel of TCR expressing cell lines.

T cell biology is integral to the study of normal immune regulation as well as cancer biology, Car-T cells, epitope specificity and antigen presentation. However, primary T cells can be difficult to propagate in culture for the length of time necessary for functional assays. In addition, primary T cells express variant T cell receptor (TCR) heterodimers that can be challenging to identify and may not be optimal for downstream studies. We sought to simplify this system using transformed T cells which can be grown in culture for extended periods of time. We engineered a floxed landing pad sequence into the safe harbor AAVS1 genetic locus using CompoZr zinc finger nucleases. Both the promoter and landing pad expression cassette are flanked by unique lox sites, allowing swapping of either the promoter and/or expression cassette as needed. We ensured that only one copy of this sequence was found within the genome to avoid any complications associated with random insertion events. We also generated a landing pad cell line null for the endogenous TCR using Cas9/CRISPR ribonucleotide complexes. Both the TCR alpha and beta loci were rendered null due to non-homologous end joining and the presence of insertions and deletions culminating in premature stop codons were genotyped using next generation sequencing. The absence of a functional TCR was validated using flow cytometry staining for surface TCR and CD3. This cell line was then used to generate a knock-in of the desired exogenous TCR heterodimer to the landing pad locus, verified using flow cytometry staining. These lines will be very useful for a multitude of studies where a researcher needs to express a gene of interest in a discrete genetic locus or wants to generate a panel of TCR expressing cell lines.