Tumor Cell Killing by T Cells: Quantifying the Impact of a CD19-BiTE Using Real-Time Cell Analysis, Flow Cytometry, and Multiplex Immunoassay
Dr. Heather Paich
Cancer immunotherapy is increasingly being evaluated as an approach to cancer treatment by harnessing the immune system to attack cancer cells. CD8+ cytotoxic T lymphocytes (CTL) directly eliminate tumor cells; the ability to correlate T cell biomarker expression/secretion with target cell killing is critical in both basic and applied studies of tumor immunology. Here, we have used a combination of impedance-based technology and flow cytometry to evaluate both the target and effector cells in a T-cell mediated B cell killing assay. Representing a promising new class of therapeutics, bispecific T-cell engagers (BiTEs) enhance the ability of CTLs to specifically recognize and eliminate tumors. Here, we investigated the capability of CD19-BiTEs to enhance the cytotoxic effects of T lymphocytes on a B-Cancer cell line, Daudi cells, in three distinct assays. Target cell death was monitored using an RTCA impedance assay and a flow cytometry-based cytolysis assay; while secretion of cytokines and cytolysis proteins were quantified in a bead-based multiplex flow cytometry assay to monitor the T cell response. The addition of CD19-BiTE enhanced T cell killing of Daudi cells up to 80% measured by either RTCA or 7-AAD staining of cell viability on the flow cytometer. Consistent with these findings, increased expression of CTL-associated proteins was observed. 7 hours after addition of effector T cells, cytokines specifically associated with CTL response such as IFN?, TNFa, and IL-2 are increased 300, 9, and 10 fold respectively. Secretion of cytolytic proteins such as sFasL, Granzyme B, and perforin also dramatically increase by 24 hours after the addition of effector T cells consistent with the CTL killing response seen. These data demonstrate that the presence of CD19-BiTE significantly enhances T cell killing of Daudi cells while increasing production of cytokines and effector molecules that mediate and sustain target cell killing. We have coupled quantitative cell killing assays with biomarker quantitation to provide an in depth view of how addition of CD19-BiTE affects T mediated killing of B cells in a single workflow. Linking cell killing data with quantitative analysis of cytokine and effector protein production allows simultaneous analysis of T cell activation and function. This workflow, which integrates both cellular and molecular phenomena, advances current methods of analysis for both basic and applied research of cancer immunotherapy
Contact: Dr. Heather Paich, email@example.com
Akadeum Life Sciences, Inc.
Microbubble-Based Cell Isolation
Leo Ostruszka, Ph.D.
Founded in 2014, Akadeum Life Sciences was established to create the simplest separation products, and to fundamentally change the way cells and other biological targets are isolated.
Akadeum’s technology overcomes limitations in current separation methods by using floating particles. These floating particles called microbubbles attach to specific cells and float these cells to the top of the sample. At the same time, untargeted cells sink to the bottom, separating the two cell types within the tube without the need for columns, magnets, or other equipment.
Contact: Leo Ostruszka, www.akadeum.com/technology
Maurice Y.F Shen, PhD
Antibodies are one of the most commonly used research reagents. However, due to their innate variability, finding the right antibody can be a challenge. Scientists devote a significant amount of time sifting through the literature to find antibodies that have been shown to work under specific experimental contexts matching their research interests. This process often takes several hours, sometimes even days, wasting valuable time that could otherwise be used more efficiently. BenchSci has created a solution to this problem by developing a machine-learning algorithm that decodes primary research papers to identify the antibody used and the associated experimental contexts in each paper. Scientists can search their protein of interest on BenchSci’s open-access online platform, and find results displayed in the form of individual figures from scientific papers in which antibodies against that protein were used. These figures are easily filterable to allow users to narrow down their search results to the antibody use cases that best fit their experimental contexts and ultimately find the best antibodies for their experiments. The BenchSci search platform allows scientists to easily identify commercially available antibodies that have been shown to work the literature in a fraction of the time, thereby facilitating the antibody search-and-purchase process.
Contact: Maurice Y.F Shen, firstname.lastname@example.org, 1 (416) 317 5798
Characterization of Non-Specific Monocyte Binding
Non-specific binding of some fluorophores like PE/Dazzle™ 594, PerCp/Cy5.5, PE/Cy5, PE/Cy7, APC/Cy7 and APC/Fire™ 750 to live monocytes has been documented, although never fully characterized. BioLegend has formulated a buffer called True-Stain Monocyte Blocker™ that effectively blocks any non-specific binding of these tandem fluorophores in antibody-based flow cytometry assays. The blocking solution does not interfere with desirable monocyte-specific antibody staining. There are many assay factors that can influence this phenomenon that are common to flow cytometry assays including stimulation of cells with PMA and the number of antibodies included in the assay.
Contact: Kelly Lundsten, email@example.com
The Celsee™ PREP platforms: From Blood to Viable Rare Individual Circulating Cells in Less Than 4 Hours
Vishal Sharma, PhD
The ability to capture rare cells of interest from heterogeneous cell suspensions or blood and to individually characterize them has, historically, been technically challenging with a significant time and financial commitment. In this presentation, Celsee Diagnostics will discuss the Celsee PREP100 and the Celsee PREP SingleCell platforms that use streamlined, simplified microfluidic technologies to detect, enrich, and characterize rare cell populations and individual cells.
The starting specimen is passed over a microfluidic chip containing tens of thousands to hundreds of thousands of individual capture wells. Cell size and morphology define which cells are captured – no fluorescent probes are needed. Three optional analysis paths exist for captured cells: on-chip immunochemistry, DNA or mRNA FISH assays, cell retrieval for off-chip characterization including PCR, NGS and culture, or individual single cells can be isolated, retrieved and analyzed.
Contact: Vishal Sharma, Ph.D., 617-272-6204; firstname.lastname@example.org
A New Standard for High Sensitivity Full Spectrum Cytometry
This presentation will provide an overview of the Cytek Aurora; a novel three-laser (up to 51 channel) flow cytometer that is uniquely suited for simple to highly complex applications. The Aurora allows detection of full emission spectra (near UV to near IR) of any fluorescent tag excited by 405, 488 and 640nm lasers. Our new multichannel semiconductor (MCS) cytometer enables high detection sensitivity in a compact optical design. Combined with an intelligent deconvolution algorithm, this technology creates a system that deftly captures light across the full emission spectrum. From resolution of multilevel hard dyed beads to stain index of fluorochrome labeled beads and cells, the Aurora meets or exceeds the performance of pricier high-end analyzers. An innovative optical detection design provides unprecedented flexibility, enabling the use of a wide array of new fluorochrome combinations without thinking about filter combinations. We show that going from simple 8-color assays to highly complex (18- to 20-color) assays in a three laser system is not only achievable, but yields high quality data with rare and dim populations easily resolved.
Go Deep for the Win: scRNA-Seq Analysis in FlowJo SeqGeq
Timothy Quinn Crawford, PhD
While fluorescent or mass cytometric technologies are currently limited to interrogating at most 40-50 protein antigens and cellular processes, single cell RNA-sequencing allows for the quantification of up to tens of thousands of RNA transcripts at single cell resolution. However, analysis and discovery using the immense amounts of data that scRNA-seq produces has been hampered by a lack of user friendly tools that do not require programming and bioinformatics experience to utilize. Herein, we describe an analysis workflow of single cell gene expression from 19 melanoma metastases in FlowJo SeqGeq, an application which provides point and click interface to bench scientists with tools to explore scRNA-seq data in real time. We demonstrate two methods to identify distinct subpopulations: 1) hierarchical gating, and 2) principle component analysis (PCA), followed by t-stochastic neighbor embedding (tSNE), then compare those populations to identify sets of differentially expressed genes, showing distinct genetic signatures for different tumors but a shared immune response between patients. Thus, we show how scRNA-seq analysis can be performed by anyone to identify gene expression networks and personalized drug targets in cancer tissues.
Contact: Timothy Quinn Crawford, email@example.com
Extracellular Vesicle analysis via immuno-magnetic separation and multiplex bead-based Flow Cytometry
The content, protein composition and surface signature of extracellular vesicles (EVs) vary greatly, and are likely to be dependent on the cellular origin, the cell’s activation status, and a variety of other parameters. Currently, markers have been identified to specifically discriminate exosomes from microvesicles, and only a select few EV surface markers have been correlated to specific cell sources. In general, the question of the heterogeneity of EV samples is rarely addressed at the experimental level, mainly due to the lack of qualified methods to analyze multiple parameters on singular EVs. The identification of specific vesicular surface markers will be of high importance to our further understanding of the molecular content and related functions of EV subsets. Miltenyi Biotec is currently applying immuno-magnetic separation and multiplex bead-based methodology to isolate and screen for new EV markers at the single vesicle level. Here, we present the technology and preliminary results obtained.
Contact: Ryan Metzler, firstname.lastname@example.org
Optimizing and validating single cell sorting of FACS using image cytometry
Tiju Theccanat (with contributions from Authors: John Tigges, Eric S. Zigon, Vasilis Toxavidis, William Rice, Leo Li-Ying Chan)
One of the major applications that is commonly performed on a fluorescence activated cell sorting (FACS) instrument is single cell sorting. Single cell sorting application is mainly used for research such as cell line development to ensure monoclonality for protein production. In addition, regenerative medicine often uses single cell sorting to study stem cell proliferation from single cell to single colony. Currently, single cell sorting is validated via light microscopy several days after initial sorting, where the cells have grown into an observable colony. However, manual observation using microscopy is highly tedious and time-consuming. Therefore, there is a need for a high-throughput, practical, and accurate detection to validate and optimize single cell sorting of FACS. In this work, we demonstrate a novel high-throughput detection method to validate and optimize single cell sorting using the Celigo Image Cytometry (Nexcelom Bioscience, Lawrence, MA). The instrument was used to image the entire well of all 96 wells on a microplate to detect a single object sorted into the well in less than 4 min. Initially, the FACS (MoFlo Astrios EQ) was used to sort single green fluorescent bead into multiple 96-well microplates in two separate experiments. The microplates were used without any buffer, thus the number and location of beads can be accurately detected. Next, the results from the two experiments showing a sort efficiency of 82 and 90% were used to optimize the FACS to increase the efficiency closer to 100%. Finally, a practical experiment was performed where GFP expressing cells were sorted into multiple 96-well plates to determine the final efficiency of sorting actual cells. After imaging and analyzing the plate data, more cells can be sorted into wells without single cell. If more than one cell exist, then this allows the users to quickly disqualify those wells and minimize the amount of reagents or time needed. The ability to rapidly detect single cell in multi-well microplates is highly important to both flow core laboratories to optimize their sorting instruments as well as to the users, who would like to confirm single cell in each well. The proposed method can highly improve the efficiency of work flow for both flow core managers and users.
Contact : Tiju Theccanat, email@example.com
Page updated on 2017-08-12 18:40:58 -0400