Anagha Divekar, Ph.D., Head of the Custom Solutions Teams with Biolegend:
“Lyophilized cells and cocktails for flow cytometry”, 15 minute product talk
Flow cytometrists performing multi-color assays often face the dilemma of whether their assays are robust and reliable. Errors can occur during cocktail preparation, cell staining, sample acquisition and data analysis and can get magnified in multi-center studies. BioLegend’s Veri-cells and lyophilized cocktail preparation can help alleviate some of these issues. Using our unique lyophilization techniques, we have been able to successfully lyophilize multi-color cocktails and human peripheral blood cells. Real time stability studies have shown that these lyophilized cocktails and cells are stable for years.
Brian Eliceiri, PhD, Professor Department of Surgery/UCSD:
“Role of Exosomes in Immune Cell Crosstalk”, 25 minute sponsored talk
Exosomes released by dendritic cells are known to modulate cell-cell immunosignaling responses by containing MHCII and relevant adapter proteins that mediate docking and activation of acceptor cells. Based on the source and maturation state, exosomes can be immunostimulatory or immunosuppressive in models of cancer, infection, tolerance, immune privilege, and arthritis. We focus on the biological activity of exosomes in models of injury to define the molecular basis for exosome release and the relevance of uniquely human genes using flow cytometry and mass spectrometry techniques.
Lisa Bellemare, BSc, Technical Applications Specialist, BD Biosciences
“New Tools for Multiparameter Flow Cytometry”, 15 minute product talk
With the advancement of technology, flow cytometry is becoming more and more powerful. We can now look at many more variables in scientific experiments with the addition of lasers, filters and dyes. However, with this increase of reagents available, creating new panels is becoming more difficult. In this short talk, we will go over the details of our newest and easy to use sorter, the FACS Melody. We will then create a nine color sorting panel using our brand new online panel design tool, the Guided Panel Solution (GPS).
Mike Stadnisky, PhD, CEO Flowjo:
“Single Cell Obsession: Unlocking n-dimensional single cell data from cytometry and single cell sequencing”, 15 minute product talk
The single cell is the basic unit of disease and the singular obsession we all share. But how do we start using emerging technologies to gain deeper insight into the 100 trillion cells of the human body, each differentiated from a single cell, which in turn has more than 10,000 transcripts and 2-4 million proteins per cubic micron? Herein, we will analyze a high-parameter flow cytometry data set using spectral compensation, data reduction, and ontology tools to name newly discovered populations and discuss a brand new collaboration and data integration tool for life science. We will also describe our work in enabling single cell sequencing analysis, showing analysis of whole melanoma metastases to compare the T-cell gene expression programs of 19 patients and reveal similarities and differences in their exhaustion. Finally, we discuss how single cell phenotyping and gene expression data can be integrated to map cellular phenotypes with 40+ proteins and 10,000 transcripts.
Qizhi Tang, PhD, Professor of Surgery, Division of Transplant Surgery, Director, Transplantation Research Laboratory, UCSF:
“Evaluation of pre-transplant immune activation and its association with rejection risk in HIV (+) kidney transplantation using Duraclone flow cytometry panels”, 25 minute sponsored talk
Purpose: HIV(+) kidney transplant recipients experience higher rates of rejection episodes when compared to HIV- recipients, with a pattern of early, aggressive rejection. One possible explanation for this pattern of rejection implicates a chronically-activated immune system in response to HIV infection, but specific markers of rejection risk have not previously been identified.
Methods: We analyzed banked, pre-transplant peripheral blood samples from HIV(+) kidney transplant recipients (HIVTR, n=82), with comparison to HIV(-) transplant recipients (n=47), HIV(+) non-transplant patients (n=25), and normal control subjects (n=25). Transplant subjects were further stratified by rejection status for analysis (HIVTR: 28 rejectors, 54 non-rejectors; HIV(-) transplant: 19 rejectors, 28 non-rejectors). We characterized the peripheral blood immune cell subsets and their expression of activation markers using three custom-designed Duraclone flow cytometric panels.
Results: HIV+ patients who subsequently experienced graft rejection have increased pre-transplant markers of immune activation. When compared to HIV(+) transplant recipients without rejection (HIVTR-NR), HIV(+) kidney rejectors (HIVTR-Rej) have decreased expression of HLA-DR on CD14+CD16+ intermediate monocytes (p=0.0042) and a trend toward decreased HLA-DR expression on classical monocytes (p=0.053) by MFI. In particular, intermediate monocytes expressing HLA-DR represent a regulatory monocyte subset, which is decreased in HIVTR-Rej patients. Additionally, HIVTR-Rej have a higher frequency of activated B cells, by percentage of cells expressing HLA-DR (p=0.034) and by HLA-DR MFI (p=0.0095). Finally, while the percentages of Tregs did not differ between groups, HIVTR-Rej were found to have a trend toward a decrease in CD39+ activated Tregs when compared to HIVTR-NR (p=0.057).
Conclusion: Our results suggest that HIV infection predisposes to chronic immune activation in the kidney transplant population, and may contribute to increased rates of rejection. HIV(+) kidney transplant recipients who subsequently experienced rejection episodes may be distinguished by pre-transplant activation status of monocyte, B cell, and Treg populations.
Vishal Sharma, PhD, Application Scientist, Celsee Diagnostics:
“The Celsee™ PREP platform: Simplified Rare Single Cell Capture and Isolation”, 15 minute product talk
The ability to capture rare cells of interest from blood, such as circulating tumor cells, and to individually characterize them has, historically, been technically challenging with a significant time and financial commitment. In this presentation, Celsee Diagnostics will outline the Celsee PREP100 and the Celsee PREP SingleCell platforms, which can also be used as a pre-enrichment complementary technique for flow cytometry, that use a simplified methodology to detect, enrich, and characterize rare circulating cells. Whole blood diluted with buffer is passed over a microfluidic chip containing tens to hundreds of thousands of individual capture wells. Cell size and morphology define which cells are captured – no fluorescent probes are needed. Two optional analysis paths exist for captured cells: on-chip assays can be performed or the cells can be retrieved for off-chip characterization. On-chip analyses include immunochemistry, DNA or mRNA FISH. Off-chip analyses of unfixed retrieved cells include PCR, NGS or culture.