Although macrophages were first described by Elie Metchnikoff in 1882, plenty of mysteries are still associated with the cell type. Indeed, while macrophages were once considered simply "garbage trucks" of the immune system due to their phagocytic property, their substantial and multifaceted contribution to immunological responses and homeostasis is becoming more apparent. Macrophages can produce a wide range of cytokines and chemokines to influence the immune response toward healing or inflammation. In as such, they possess a great deal of plasticity to respond with either pro- or anti-inflammatory signals depending on the environmental milieu. Moreover, researchers are beginning to turn to macrophages to assist chimeric antigen receptor (CAR) T cells in various immunotherapies. This webinar is presented by Anne Lodge, Chief Scientific Officer of Astarte Biologics.

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The World of Macrophages: How to Harness Them for Research

Functional imaging is a rapidly growing field key to driving new understanding in biology. Insights into the function and interaction of molecules are the key to reveal the underlying cellular mechanisms. In this context, fluorescence lifetime imaging (FLIM) is a powerful tool, providing valuable information beyond spectral imaging. FLIM is immune to concentration artefacts and sensitive to molecular environment such has pH changes, ion concentrations, and more. Förster Resonance Energy Transfer (FRET) is an example of molecular environmental changes. The donor lifetime is shortened by the presence of the acceptor. FRET experiments thus benefit from FLIM information. The FRET-FLIM readout is independent of the donor or acceptor concentration making it the quantification assay of choice. Recently, FRET has been exploited to engineer sensors (FRET biosensors). FRET signal changes in these biosensors correspond to either binding or release of a ligand. How this type of readout can help uncover biological mechanisms was recently shown in a Nature Immunology article (Anzilotti 2019). The approach used in this article, and described in this webinar, opens the field to imaging in a range of situations in primary cells, where sensitivity and the need to avoid damaging the cells are both paramount.

Basics of FLIM and Applications to FRET Biosensors

In this tutorial on confocal imaging, you will learn how you can: - Capture weaker signals—and still get sound, reproducible data. - Reach faster volumetric imaging without sacrificing resolution - Increase data throughput for your imaging needs Learn about the new Multiplex mode for parallel pixel acquisition with the ZEISS LSM 9 family and Airyscan 2. You can now acquire up to 8 superresolution image lines with high signal-to-noise rate in a single sweep. Capture dynamic processes, cellular signaling, molecular trafficking, and diffusion events with real-time superresolution and high SNR. The new Multiplex mode for Airyscan 2 uses smart illumination and detection schemes for parallel pixel acquisition on a confocal microscope. Scientists can now capture weaker signals, keep their context, and get statistically sound data. Extending Airyscan imaging to larger model systems with lower expression levels, the new Multiplex mode increases acquisition speeds even further. You get superresolution and a 4 times higher SNR compared to traditional confocals. This novel concept allows rapid volumetric imaging with unprecedented resolution beyond what is available in traditional confocal systems today. Airyscan 2 provides new data handling concepts, providing 6.6 times smaller data sizes and 5 times faster image reconstruction times. Further, optimized real time acquisition strategies employed with the LSM 9 family enable faster scan speeds for Airyscan 2, allowing higher data throughput.

Adding Multiplex Factor to Confocal Imaging with ZEISS LSM 9

In this webinar, you will learn: - The complete workflow for in situ cryo-electron tomography - How subtomogram averaging within the cell yields native-state structures of macromolecular complexes (e.g., the asymmetric and dilated nuclear pore of algae) - How mapping these structures back into the native cellular environment reveals new molecular interactions that are only accessible by this technique (e.g., the binding of cargo to COPI-coated Golgi membranes and the tethering of proteasomes to the nuclear pore). Cryo-electron tomography can visualize macromolecular structures in situ, inside the cell. Vitreous frozen cells are first thinned with a focused ion beam and then imaged in three dimensions using a transmission electron microscope. This transformative method has the power to revolutionize our understanding of cell biology, revealing native cellular architecture with molecular clarity.

Cryo-electron Tomography for Cell Biology

In this webinar, you will learn: - How structural biology can change the face of your research - The biological challenges of cancer research - Advantages of cryo-EM for your research Covering both biology and methodology, this webinar will explain how single-particle cryo-electron microscopy enables us to gain insight into cancer development through the detailed analysis of molecular structure. In single-particle cryo-EM, hundreds of thousands of images formed by electron scattering of individual molecules or complexes are analyzed to derive their three-dimensional structure. Technological and computational advances have dramatically transformed the field of cryo-EM in the past years, enabling structural insights at near-atomic resolution into assemblies that had not been tractable using any other structural biology technique. Consequently, cryo-EM has become a mainstream method structural biology, with a multitude of new facilities and research groups being established all over the world within just a couple of years. This webinar will address the roles that structural biology has been playing in cancer research, uncovering cellular processes involved in cancer development and protection, and guiding drug discovery efforts. The biological challenges of cancer research will be discussed, as well as the unique strengths of cryo-EM as an experimental approach towards these questions, briefly covering the methodology and procedures in sample preparation and data processing. We will illustrate these aspects with some of the latest research from the laboratory of Eva Nogales at UC Berkeley and assess the promises and challenges of cryo-EM in our fight against cancer.

Cryo-Electron Microscopy and the Complexity of Cancer

Your life sciences research often requires you to measure, quantify and understand the finest details and sub-cellular structures of your sample. You may be working with tissue, bacteria, organoids, neurons, living or fixed -cells and many different labels. In this webinar, we will explain how Elyra 7 with Lattice SIM takes you beyond the diffraction limit of conventional microscopy to image your samples with superresolution. Learn how to examine the fastest processes in living samples – in large fields of view, in 3D, over long time periods, and with multiple colors. The new Lattice SIM technology of Elyra 7 brings structured illumination microscopy (SIM) to a new level. Groundbreaking light efficiency gives you gentle superresolution imaging with incredibly high speed – at 255 fps you will get your data faster than ever before. See how Elyra 7 lets you combine Lattice SIM with single molecule localization microscopy (SMLM) for techniques such as PALM, dSTORM and PAINT. Choose freely among your labels when imaging with resolutions down to 20 nm laterally. High power laser lines allow you to image your sample with ease, from green to far red. Elyra 7 is also very flexible: you can employ a wealth of contrasting techniques and combine them with optical sectioning. The new Apotome mode gives you superfast optical sectioning of your 3D samples. All that, plus Elyra 7 works seamlessly with your ZEISS SEMs in a correlative workflow.

Fast superresolution microscopy – with Lattice SIM

Array tomography (AT) is a 3D image reconstruction technique for high resolution, quantitative analysis of biological structures. For optimal results, ultrathin and ordered sections are an absolute requirement. In this webinar you will get tips and tricks to optimize the workflow of your array tomography: - Fast and precise trimming of the sample block-face - Adhesion of a single section to create ribbons - Automated serial sectioning with the ARTOS 3D ultramicrotome - Acquisition and processing of a 3D SEM dataset - Segmentation and 3D reconstruction of cells - Interpretation of 3D reconstructions

Array Tomography for SEM 3D Reconstruction

In this webinar you will learn: - Improved vitrification: Specimen vitrification without synchronisation fluid - Vitrification strategies: Optimized freezing for different techniques - Light and electrical stimulation: Dissect cellular processes with millisecond precision - A look into the future: freezing of crystals Plunge freezing and cryo imaging of proteins and complexes have revealed new details in understanding the machinery of the cell and how molecules are involved in cellular processes. However, most eukaryotic cells and tissue samples cannot be plunge frozen because of the rapid decay of the cooling rate within the sample during freezing. High pressure freezing, on the other hand, is currently the main approach to vitrify larger samples (up to 200 µm) and to capture the intrinsic changes in fine structure or cellular dynamics. To further improve its cryo solutions, Leica developed a new cryo platform: the EM ICE. This new generation cryo platform combines speed, reliability and flexibility to facilitate research in various scientific fields. The EM ICE allows users to freeze samples within milliseconds and even permits the combination of high pressure freezing with optogenetics and electrophysiology.

Expanding the limits of EM sample prep with ICE

Join Dr. Jason Reed as he describes a novel method by which endonuclease-inhibited Cas9 can be employed as a programmable biomarker in high-speed atomic force microscopy (HS-AFM) imaging. In this webinar, you will learn: 1. How CRISPR/Cas9 can be used to "flag" alterations and mutations in DNA, rather than cut it 2. How pairing atomic force microscopy (AFM) with optical equipment found in DVD players can be used to map DNA at a faster rate than traditional DNA sequencing 3. Applications of this technology, particularly in relation to discovering and diagnosing genetic diseases Since the diameter of the Cas9 molecule is greater than that of DNA, they are easy to locate along the DNA strand. Taking advantage of this, Jason's lab reported approximately 90% Cas9 binding accuracy to DNA molecules under optimized conditions. The alignment of single-molecule maps with nanoscale resolution becomes far more computationally straightforward than if labels are localized with multi-kb ambiguity. This process yields reduced processing time and cost for assembling a consensus map. Given its single-molecule sensitivity, approximately 15 bp accuracy, and no amplification requirement, Dr. Reed's novel method is amenable to small sample sizes. This proves to be an advantage in clinical situations where obtaining the almost 10 μg of DNA required for single-molecule sequencing is extremely difficult—if not impossible!

Using CRISPR/Cas9 to detect DNA sequences with AFM

Join Theo Roth as he describes his lab's novel CRISPR-Cas9 genome-targeting system that does not require viral vectors to modify T cell genomes, but instead focuses on HDR. This allows rapid and efficient insertion of large DNA sequences at specific sites in the genomes of primary human T cells, and permits individual or multiplexed modification of endogenous genes. Importantly, avoiding the use of viral vectors will result in accelerated research and clinical applications, reduce experimental cost, and improve safety. In this webinar, you will learn: - The advantages of using HDR versus recombinant viral vectors when modifying T cell genomes - How long double-stranded and single-stranded DNA can serve as a non-viral HDR template - A novel method that allows for the insertion of large DNA sequences (>1Kb) without a virus! Current efforts at reprogramming T cells for therapeutic purposes rely on using recombinant viral vectors. Unfortunately, viral vectors do not target transgenes to specific genomic sites. Moreover, the manufacturing and testing of effective viral vectors is often a lengthy and expensive process, which slows research progress and clinical use. However, recent studies have shown that re-engineering T cells in a specific and efficient manner is possible using homology-directed repair (HDR).

How to Use CRISPR to Accelerate Cancer Therapies

Join us in this webinar as Dr. Victoria Doronina helps you determine the quality of your nucleic acids. In this webinar you will learn: - How to choose the best method to extract your nucleic acids - Which method you should chose to determine nucleic acid quality - How to avoid common pitfalls for both extraction and quality control Virtually all experiments in the molecular biology lab require high quality, pure nucleic acids as a starting material. This sounds simple enough. However, as always, the trouble is in the details. There are several methods to isolate your nucleic acids. But which one is best for your experiment? How do you determine nucleic acid quality? And why does it seem to not be working? Watch this webinar to find out the answers. Victoria will breakdown the nucleic acid purification process and how to match the right method to the right type of nucleic acid. She will also show you how to avoid common pitfalls and contaminants in isolation and quality control. This webinar is essential for anyone in a molecular biology lab who wants better results in downstream experiments!

Nucleic acids 101: Confirming Their Quality

In this webinar, you will learn: - How to best prepare your specimen for live cell isolation by laser microdissection - How to optimize your laser microdissection workflow - How to avoid common pitfalls of this technique Laser microdissection is a tool for the isolation of homogenous cell populations from their native niches in tissues to downstream molecular assays. Beside its routine use for fixed tissue sections, laser microdissection may be applied for live cell isolation. Unlike other well-established and widely used techniques for live cell isolation and single cell cloning—such as FACS, MACS, cloning by limited dilution, and so on—laser microdissection allows for capturing live cells and cell colonies without their detachment from the carrier. In other words, there is no need to prepare a single cell suspension before the isolation procedure using mechanical and enzymatic dissociation, which can affect cell fate after plating. This feature of laser microdissection is desirable for stem cell research. We established a simple strategy for the efficient live cell isolation using the Leica Laser Microdissection platform. We were able to demonstrate not only colony formation from the isolated samples containing live cells, but also single cell cloning. In this webinar, specimen preparation, laser adjustment, overall workflow, and limitations on live cell isolation by laser microdissection are discussed.

Live Cell Isolation by Laser Microdissection

In this webinar, you will learn: - How to overcome issues with stage drift and crosstalk between channels - How to obtain reliable images and reproducible quantification results - How to proceed with advanced image analysis after restoring images During fluorescence image acquisition, many experimental uncertainties are introduced that affect the correct object interpretation and analysis. The blurring and the noise implicit in the image formation are two of the largest sources of experimental trouble. Additional aberrations, such as stage drift, and crosstalk and chromatic aberration between channels, can also affect the imaging. Huygens image deconvolution and restoration is a proven method to revert these issues and recover a more realistic representation of the original object. After restoration of the image, you can proceed with the advanced Huygens analysis options for colocalization, object measurements, and tracking. This webinar will illustrate how you can make optimal use of the complete Huygens workflow to obtain reliable images and reproducible quantification results, focusing on the advanced analysis options offered by the Huygens software.

Reliable quantification of fluorescence images

Join us in this webinar featuring Dr. Omonse Talton who will guide you through developing a "fool proof" enzyme-linked immunosorbent assay (ELISA). In this webinar, you will learn: - When and why you should use the different types of ELISAs– direct, indirect, sandwich, competitive/inhibition ELISA - Major considerations for developing your ELISA - Tried and tested tips for you to perform a successful ELISA The ELISA is one of (if not the most) common techniques in biology and biochemistry laboratories. You can use an ELISA to detect minute amounts of protein for medical diagnostics, for testing food for common allergens, and in toxicology screens screen for certain drugs. While the overall premise of an ELISA is simple, as with any assay, its success hinges on the experimental set up. In this webinar, Omonse will give you a crash course on that experimental set up—from start to finish. She will discuss the different types of ELISAs, the major considerations in development of the assay, and troubleshooting—including tips and tricks. With this webinar, you will be able to ensure that your ELISA provides reproducible and publishable results!

Crash Course: Developing a Fool-Proof ELISA

In this webinar, Dr. Stephen Pettitt explains how he applies genome-wide targeted mutagenesis screens to elucidate the genetic basis of drug resistance. Using mouse and breast cancer cell lines, Dr. Pettitt’s team developed a targeted, genome-wide mutagenesis screen to identify mutations responsible for resistance to the potent PARP inhibitor talazoparib (BMN 673). The screen yielded one particularly interesting point mutation in the PARP1 gene. This mutation disrupted the ability of PARP1 to bind DNA, demonstrating that DNA binding is necessary for the action of talazoparib. Dr. Pettitt will describe how he then employed a high-density, focused sgRNA library targeting PARP1 to generate further mutants that he used to elucidate details of the structure-function relationships of PARP1. This research is not only important for unravelling the mechanisms underlying drug resistance, but it may improve future treatment plans for cancer patients. In this webinar, you will learn: - How to use genome-wide CRISPR screening for mutant discovery - How to create a highly diverse, sgRNA library from Twist Bioscience for targeted, subtle mutations - How knowledge of the structure-function relationships of PARP1 mutants can inform treatment of cancer patients with these drugs

Discovering PARP inhibitor resistance mechanisms with CRISPR

Bioscience Research Tips and Advice Webinars from Bitesize Bio

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