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Session Type: Workshop
Name It! Store It! Protect It! Identifying Data Management Protocols for Core FacilitiesThis 75 minute session is jam packed with the foundation of basic principles for core directors and administrators to gain a 101 understanding of file naming, data storage, and data management. Four speakers will be presenting on some best practices of how to name your files, how to manage the storage of data – especially large file sizes – and how to protect research data. We will be providing a short discussion and handout about NIH expectations and policies on data management. This is a jam packed session that will help core directors of all scientific disciplines and administrators understand the basics (and challenges) of managing data in a research environment. It is our hope to demonstrate some of the best practices that work well for our institutions, to help others to overcome some of the many challenges that we all face when it comes to managing data.
Session Type: Roundtable
iABRF Roundtable Discussion: Advancing Core Technology Sciences and Communication through Establishment of a Worldwide Core FederationCore facility laboratories are critical to the research mission of the institute and community they serve. Many core focused associations, societies, and workshops are established worldwide with similar goals to advance the core sciences. ABRF has an interest to identify ways that the ABRF could best encourage the establishment and growth of core facility organizations and meetings around the world and on ways that the ABRF could best interact and coordinate with such organizations and meetings. Towards this end, the International ABRF Committee supports to work collaboratively with existing and emerging worldwide groups towards the establishment of a worldwide federation of core facility interest groups, in which the core facility interest groups from different countries are all treated and represented as equals. The mission of this federation would be to ensure international communication and collaboration around core facility matters. The benefit to the ABRF would be (1) increasing ABRF participation in efforts to advance core science and administration world-wide, and (2) to coordinate with and collaborate with core facility interest groups around the world. This session will have representation from numerous other core interest groups to help ascertain the common goals, their mandate, and how we might be able to form and interact under such a worldwide federation of core facility interest groups. The session will encourage attendee interaction and presented in a roundtable format.
Session Type: Poster Session
Channel-free dead cell exclusion in FACS/flow cytometry (or "n+1 into n" does go)Dead cell exclusion is a common requirement for flow cytometry on fresh, unfixed samples. In multi-colour phenotyping this occupies one or more channels reducing dimensionality and options for antibody-chromophore pairings. This limitation is obvious but further exacerbated if dead cell exclusion is identified or demanded retrospectively. Then, panel re-formatting into two tubes (linking antigens increase cost) or transfer to a higher dimensionality platform may be required. Importantly, additional demands are then placed on core facility capacity / throughput. Meanwhile, dead cell exclusion with DAPI occupies channels valuable for the new violet-excited chromophores and, alternatively, propidium iodide occludes R-PE, a bright and widely conjugated chromophore. A novel yet simple remedy is achieved using DRAQ7, an anthraquinone-based far-red fluorescing viability probe, validated in flow cytometry and fluorescence microscopy. Its absorbance spectrum and practice show DRAQ7 can be detected using excitation wavelengths of blue to red lasers; potentially then by two lasers in one analysis. This unique property locates DRAQ7+ events in a unique region of bivariate plots for available far-red/NIR channels. A “live” gate is then set on the preferred bivariate plot for that experimental set-up, excluding dead cells in all channels. In one example, a high-throughput flow cytometry platform limited to 4-channels was hampered by use of propidium iodide reducing antigen phenotyping to 3 channels, bleed through to other channels, and reducing from 16 to 8 the finite number of phenotypes that might be elicited from the antigen analysis. Substitution with DRAQ7 re-enabled use of all 4 channels for antigen expression analysis. It is noteworthy that there is no need for compensation and that this simple method can be applied retrospectively (e.g. on reviewers' request) without disturbing antibody panel design. Due to DRAQ7’s previously demonstrated ultra-low toxicity this method can also be used in cell sorting.
Session Type: Scientific Session
3D Printing for the CoreThree-Dimensional (3D) printing is a disruptive technology that puts the prototyping and manufacturing process directly into the hands of the inventor. Rapid prototyping and the additive manufacturing process promise to reshape the laboratory and shift the learning paradigm in the classroom. We will explore the basic concepts of 3D printing, rapid prototyping and 3D replication, as a core asset. We will also discuss specialized materials, where to find resources, and innovations in the field of 3D printing. This workshop will be in visual presentation format with a step-by-step walkthrough of the 3D printing process, with demonstration.
Highly multiplexed simultaneous measurement of cell-surface proteins and the transcriptome in single cells.Large-scale, unbiased identification of distinct cell types in complex cell mixtures has been enabled by recent advances in high-throughput single-cell transcriptomics. However, these methods are unable to provide additional phenotypic information, such as the protein levels of well-established cell surface markers. Current approaches to simultaneously detect and/or measure transcripts and proteins in single cells are based on 1) indexed cell sorting in combination with RNA-sequencing or 2) proximity ligation/extension assay in combination with digital PCR. These assays are limited in scale and/or can only profile a few genes and proteins in parallel. To overcome these limitations, we have devised a method, Cellular Indexing of Transcriptome and Epitopes by sequencing (CITE-seq), that combines unbiased genome-wide expression profiling with the measurement of specific protein markers in thousands of single cells using droplet microfluidics. We conjugate monoclonal antibodies to oligonucleotides containing unique antibody identifier sequences. We then label a cell suspension with DNA-barcoded antibodies and single cells are subsequently encapsulated into nanoliter-sized aqueous droplets in a microfluidic apparatus. In each droplet, antibody and cDNA molecules are indexed with the same unique barcode and are converted into libraries that are amplified independently and mixed in appropriate proportions for sequencing in the same lane. In proof-of-principle experiments using a suspension of mixed human and mouse cells and established high-throughput single cell sequencing protocols, we unambiguously identify human and mouse cells based on their species-specific cell surface proteins and independently on their transcriptome. We then use CITE-seq to classify cells in the immune system, which has been extensively characterized on the level of cell surface marker expression. We show that we are able to achieve better resolution of cell types by adding an extra dimension to the data. CITE-seq allows in-depth characterization of single cells by simultaneous measurement of gene-expression levels and cell-surface proteins, is highly scalable, only limited by the number of specific antibodies that are available.
Network-based prioritization of de novo mutations in Autism and EpilepsyObjective: Up to 30% of patients with autism have epilepsy. The genetic basis of autism and epilepsy has not been well characterized. We analyzed data from whole exome sequencing (WES) on individuals with autism and epilepsy to prioritize pathogenic rare de novo variants. Methods: Detailed clinical and demographic data of over 90 individuals with autism who subsequently developed epilepsy were collected. Whole exome sequencing was performed to identify rare de novo variants. We developed a model to predict genes at the interface of autism and epilepsy and performed network analysis of signaling pathways to identify variants for validation by Sanger sequencing. Results: Rare de novo variants were identified in 73 of 92 cases (79%). Our model predicted 9 genes, termed “hot genes”, at the interface of both disorders (PAK1, SCN5A, NTRK1, GRIN2B, HDAC4, GRIN2A, MYH2, NRXN1). Network analysis identified the MAP kinase and calcium signaling pathways as being over-represented in our cohort. Sanger sequencing of selected de novo variants in 23 cases was performed with a 98% validation rate. The most commonly mutated gene in our cohort was GRIN2A which was found in 3 individuals with generalized epilepsy. We identified likely pathogenic de novo variants in several genes not previously implicated in autism or epilepsy including hot genes MYH2, and PAK1 as well as MAPK/Ca signaling genes HSPA1B, FGFR1, PPP3CA and SLC8A1. The MAPK pathway was associated with severe intellectual disability in our cohort. Interpretation: Rare de novo variants likely contribute to the development of autism and epilepsy. A significant portion of our cohort had a positive family history of autism, epilepsy, psychiatric disease or autoimmune disorders suggesting the genetics of autism and epilepsy are complex with contributions from common and rare inherited variants as well as disease causing de novo variants.
Precision Immunology Through Deeper Single Cell ProfilingThree trends have dominated biomedical research over the last decade. The first, the NIH Roadmap’s Single Cell Analysis Program, was founded on the principle that cells are extremely heterogenous, and that this heterogeneity is important in health and disease. For this reason, cells must be characterized individually, rather than by insensitive and misleading analysis of bulk cell populations. This trend renewed appreciation for cellular heterogeneity, and incited a revolution of new technologies that could comprehensively analyze single cells (the second trend, deep profiling). Finally, a third biomedical research trend was sparked by President Obama’s Precision Medicine Initiative, which aims to define genomic and proteomic differences between patient groups, and use this information to inform treatment decisions. In this talk, I will discuss my work at the intersection of these three trends, and demonstrate the value of new technologies for comprehensive and complete cellular analysis. I will provide examples of how deep knowledge about immune responses can be attained, using examples drawn from our recent work in HIV vaccine settings, immunotherapy, and fundamental immunology. This talk will highlight our work developing 30 parameter flow cytometry, single cell RNA sequencing, and new bioinformatic tools and include some discussion of how microfluidics and nanotechnologies can fit into a pipeline that includes the above technologies.
A method for in situ multiplexed targeted protein profiling of circulating tumor cellsCancer evolves in the patient from initiation to widespread metastatic disease through a series of changes through both natural progression and response to treatment pressure. This spatio-temporal evolution, while influenced by many factors, has to be characterized at the genomic and proteomic levels to develop and implement patient specific treatment approaches. While DNA and RNA based approaches are in routine research use in single cell biology today, there is a need for protein analysis of tumor cells in patient’s blood. The described method is an extension of the HD-SCA (High definition single cell analysis) assay liquid biopsy approach where rare circulating tumor cells (CTCs) are identified using 4 color fluorescence staining, and further characterized by Imaging Mass Cytometry (IMC-CyTOF, Fluidigm corp) enabling simultaneous targeted proteomic analysis for a panel of up to 38 markers. A panel of cancer and blood cell relevant protein markers and corresponding antibodies have been identified. Each antibody has been conjugated to a lanthanide metal isotope required for IMC-CyTOF analysis and evaluated using various cell lines spiked in leukocytes from normal blood and plated on glass slides. All spiked cells were identified with the HD-SCA assay and a subset was further stained, relocated and analyzed with IMC for the feasibility of this approach for multiplexed protein profiling of CTCs on a single-cell level. Surrounding leukocytes are used both as internal quality control and for data normalization purposes, enabling reproducible scoring of markers against background levels. We believe that the described method will allow the fluid biopsy to take an important leap towards personalized medicine in clinical practice.
Session Type: Platinum Presentation
The Road NOT Taken – A MoFlo Astrios Sort Logic Path to DiscoveryIt is widely known that cell sorters provide rapid quantification and cell purification by incorporating multi-parametric approaches using fluorescent dyes and labels. Contemporary researchers incorporate transgenic fluorescent proteins for identification and use cell sorting as a pass through technology to capture cells for downstream studies. We’d all agree that knowing how to identify the target and learning how the cell sorter prioritizes cell capture is critical to a researcher’s success yet empirical testing of how sort logic impacts outcome is rarely a specific aim. As such, many investigators continue with the status quo when at the flow core – they use the same sorter and sort conditions – the stress of getting to a triplicate is far too great to spur exploration. In this presentation, I will share a story of how an investigator improved his RNAseq data by utilizing MoFlo Astrios sort modes and gate logic.