Abstracts

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Vera Tang,  “Small particle flow cytometry: Size matters, but it’s complicated”

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The application of flow cytometry (FCM) in the analysis of small biological particles such as extracellular vesicles and viruses has been steadily increasing in the recent years. The large majority of small particle FCM utilizes commercially available flow cytometers designed for analysis of cells, which are much larger in size with proportionately higher levels of antigen expression. Although it is possible to detect small particles using these instruments, they are functioning at the limit of detection to do so. Commercial flow cytometers have a wide range of optical configurations. This talk will focus on outlining best practices and methods for analysis of small particles by FCM that will allow for data optimization and standardized reporting across different instruments.

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Evan Jellison, “Looking at Flow Cytometry Biosafety with 2020 Vision”

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This year has been unprecedented in terms of how flow cytometrists, especially those working in shared resource labs, have had to reconsider their working environment. While many cytometrists are accustomed to working with biohazards of various classifications in a research or clinical setting, even those who normally work in a “clean” environment have had to manage the potential for a new biohazard in their laboratory: the SARS-COV-2 infected user. Fortunately, many of the procedures and environmental controls necessary to mitigate the risk of working under this new pandemic threat have already been established by international organizations including ISAC, ABSA, CDC, WHO, as well as institutional biosafety committees around the world. In this talk we aim to examine some of the procedures and recommendations that have been made and, more importantly, how individuals can evaluate their own procedures; because ultimately, it doesn’t matter what anyone else does, it only matters what you do with your users in your setting.

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Marcela Carmona, “Molecular Signatures of Motor Cortical Diversity During Learning”

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For humans, and most animals, movement is required to interact with the surrounding world. However, as is especially apparent when observing babies of many species, most movements must first be learned for their precise execution. The motor system, encompassing several brain areas, the spinal cord, and its interaction with skeletal muscle orchestrates movement and has been well delineated, but what role these players have during motor learning is still unclear. One region, the primary motor cortex (M1) is of special interest as it appears to be essential for the learning of many skilled motor tasks. Recent studies have placed emphasis on identifying the circuits involved and characterizing motor cortical cell dynamics during this process. This has, however, left the cellular determinants of this process unexplored. Do different genetically defined cell types in M1 play different roles during motor learning? Is their involvement during this process static from early to late learning of does it change over time? Are there molecular and cellular changes within these cell types that are necessary and important in achieving the goal of learning a new motor skill? The ability to isolate active cells during the motor learning is a key requirement to answer these questions. We have, therefore, developed a strategy to isolate active cells using flow cytometric electrostatic droplet sorting by marking them with the calcium dependent ratiometric photoconvertible system, CaMPARI. Subsequent single-cell RNA sequencing of sorted cells has allowed us to transcriptionally profile active cells and to begin to determine which cell types are engaged throughout learning and what cell state changes they exhibit.

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Florian Mair, “Defining the phenotype and function of antigen-presenting cells and T cells in the human tumor microenvironment using multi-omic single cell techniques"

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Studies in the past decade have highlighted the distinct functional properties of immune cell subsets that reside in non-lymphoid tissues. Most notably, tissue-resident memory T cells (TRM) have been well characterized as a critical population for steady-state tissue immunity and during anti-tumor immune responses. Contrary, relatively little is known about myeloid antigen-presenting cells (APCs) in human tissues, namely dendritic cells (DCs) and macrophages, which are critical for shaping local T cell function.
We used a combination of single cell RNA sequencing (scRNA-seq) and 30-parameter flow cytometry to comprehensively study the APC compartment in human oral squamous cell carcinoma (SCC) relative to oral mucosal reference biopsies. Analysis of the cytometry data using a novel method termed FAUST (Full Annotation Using Shape-constrained Trees) allowed automated discovery of tumor specific phenotypes. Combined with our transcriptomic analysis, our data reveal that mucosa-resident APCs show tissue-specific specialization, including the expression of key co-regulatory receptors and T cell attracting chemokines such as CXCL16. Analysis of T cell-APC interactions using NicheNet revealed CD4 and CD8 T cell specific ligand-receptor networks. Our ongoing efforts focus on the validation of these interaction patterns using the combination of 30-parameter cell sorting and targeted transcriptomics experiments.
Overall, our data provide insight into the heterogeneity of human APCs in mucosal tissues, which changes occur in the tumor microenvironment, and how these APCs can influence the function of tumor-infiltrating T cells.

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Tomas Kalina, “Standardization of cytometry and antibody validation”

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There is an agreement in the field that interlaboratory reproducibility of flow cytometry measurements as well as whole studies might be improved by a consensual use of a methodological approach. Typically, a consensus is made on the crucial markers needed in the immunostaining panel, sometimes on the particular fluorochrome conjugates and rarely on a complete set of methods for sample preparation. The term "standardization" is used to describe the complete set of methodical steps, while "harmonization" is used for partial agreement on the method. Standardization can provide a platform for improved reproducibility of cytometry results over prolonged periods of time, across different sites and across different instruments. The different standardized approaches can and in fact should serve as benchmarking reference tools for the development of future flow cytometry studies. The cornerstone of each flow cytometry experiment is the reagent(s) used. Typically, these reagents are monoclonal antibodies conjugated to fluorochromes. Their quality performance is an absolute requirement for reproducible flow cytometry experiments. While there is an enormous body of antibody reagents available, there is still a lack of consensus about which criteria should be evaluated to select antibody reagents with the proper performance, how to validate antibody reagents for flow cytometry, and how to interpret the validation results. This presentation summarizes the co-development of cytometry, antibody development and validation strategies. It discusses the diverse issues of the specificity, cross-reactivity, epitope, titration, and reproducibility features of antibody reagents. It highlights the efforts of Human Cell Differentiation Molecules in the organization of the CD nomenclature through HLDA workshops.

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Nicole Poulton, “Exploring Marine Environments and Monitoring Microbial Patterns using Flow Cytometry”

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The ability to identify environmental or climatic changes requires monitoring programs that measure well-defined variables for sustained periods of time. In order to understand differences or anomalies on annual to decadal scales, especially those caused by anthropogenic sources, a large number of observations are required for statistical significance. Marine microbes are a key component of the diversity and functioning of coastal marine ecosystems and long-term time series observations are essential to determine ecological responses to climate variability and change. These microbes (archaea, bacteria and protists) comprise a vast majority of biological diversity on our planet and perform a significant portion of the global biogeochemical processing, such as carbon and nitrogen fixation, organic material degradation, and recycling of the life-sustaining elements phosphorus, sulfur, and iron. This talk will explore the use of flow cytometry in marine microbial studies and findings from a long-term time series located in the Gulf of Maine for the past 20 years. Seasonal variability of marine microbial groups may be due to changes in local physical forcing or climate warming. These data will provide a basis for assessing, modeling, and predicting effects of changing environmental conditions.

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Rachael Sheridan, “Approaches for Handling Challenging Samples”

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Shared Resource Flow Cytometry labs support a wide variety of research programs. In addition to routine samples, facility users often bring challenging and unique samples. In the biomedical context, these samples may include subcellular organelles such as nuclei, debris-ridden tissue preps, and samples with very high levels of cell death. This talk will cover some useful approaches and tips for working with these types of samples.

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