2nd Annual Conference
2012
The Second Annual Imaging and Flow Cytometry Research Day was held on Wednesday, September 19th and was a huge success! Over 175 registrants and 19 vendors participated in the event.
Below are the featured presentations and abstracts from the 2012 Imaging and Flow Cytometry Research Day. Please click on the links to watch the full presentations and feel free to contact the presenters with any questions.
Title: "Dissecting Cross-Presentation of Cell-Associated Antigens in Dendritic Cells by Imaging"
Edith Jansen, PhD, Cincinnati Children's Hospital Medical Center
Abstract: Every day millions of cells die in the body, producing cellular corpses and material that must be disposed of. The sensing and clearance of dying (apoptotic) cells is generally considered to be a non-inflammatory or even tolerizing process. The prevailing view has been that apoptotic cells generated by normal tissue turnover are captured by dendritic cells (DCs) that migrate to local lymph nodes, where they induce T cell tolerance, T cell anergy, or T cell deletion of self-reactive T cells in order to maintain tissue homeostasis and prevent autoimmunity in the host. Cross-presentation of cell-associated antigens (Ag) plays an important role in the induction of autoimmune diseases, anti-tumor responses, and transplant rejection. While several DC populations can induce pro-inflammatory CD8+ T cell responses to cell-associated Ag during infection, in the absence of infection, cross-priming of naïve CD8+ T cells is highly restricted. Using a combination of novel imaging approaches we found that cross-priming DCs display a distinct phenotype in cell-associated Ag uptake, endosomal/lysosomal trafficking, lysosomal acidification, and Ag persistence compared to non-cross-priming DC populations. Subtle differences in internal processing pathways and their signaling sequelae significantly affect the duration of Ag cross-presentation and cytokine production by DCs, thereby shaping the ensuing CD8+ T cell response. Our studies demonstrate that the combining of imaging techniques provides increased mechanistic insight into cross-presentation pathways and may identify new therapeutic targets for autoimmune disease, transplantation, and cancer.
Title: "Cytometry of DNA Damage Signaling"
Zibgniew Darzynkiewicz, MD, PhD, New York Medical College
Abstract: Briefly reviewed will be molecular mechanisms of DNA damage signaling that can be assessed in individual cells by flow- and image-assisted cytometry and correlated with the cell cycle phase and induction of apoptosis or cell senescence. Presented will be examples of DNA damage induced by cell treatment with UV light, DNA topoisomerase I or II inhibitors, cisplatin, tobacco smoke, and by exogenous and endogenous oxidants. Chromatin relaxation (decondensation), an early event of DDR chromatin that involves modification of high mobility group proteins (HMGs) and histone H1, can be detected by analysis of the susceptibility of DNA in situ to denaturation using the metachromatic fluorochrome acridine orange. Translocation of the MRN complex consisting of Meiotic Recombination 11 Homolog A (Mre11), Rad50 homolog and Nijmegen Breakage Syndrome 1 (NMR1) into DNA damage sites can be assessed by laser scanning cytometry as the increase in the intensity of maximal pixel as well as integral value of Mre11 immunofluorescence in the nucleus. Examples of cytometric detection of histone H2AX phosphorylation on Ser139 (γH2AX) and activation of Ataxia telangiectasia mutated (ATM), and Check 2 (Chk2) protein kinases using phospho-specific Abs targeting Ser1981 and Thr68 of these proteins, respectively also will be presented. Discussed will be approaches to correlate activation of ATM and Chk2 with phosphorylation of p53 on Ser15 and histone H2AX on Ser139 as well as with cell cycle phase and DNA replication. A combination of EdU incorporation detected by “click chemistry” and visualization of DNA double strand breaks reported by the presence of γH2AX foci, detected by laser scanning cytometry or confocal microscopy, makes it possible to explore the role of DNA replication in the induction of DNA damage by UV, oxidative stress or DNA topoisomerase I and II inhibitors. The capability of laser scanning cytometry to quantify individual foci of phosphorylated H2AX and/or ATM that provides more dependable assessment of the presence of DNA double-strand breaks is outlined.
Title: "More Lasers, More Colors, More Options: Maximum Flexibility for Flow Cytometric Analysis"
Bill Telford PhD, National Institute of Health
Abstract: In the last ten years we have seen a dramatic jump in the data collection and analysis capabilities of flow cytometers. Multilaser instruments are now the norm, with at least three lasers often being the minimum for comprehensive analysis. Simultaneous analysis of eight or more fluorescent parameters is now routine. The variety of fluorescent probes available for multicolor labeling has greatly expanded, and the software allowing analysis of high-dimensional cytometry data is increasing in capability and complexity. Imaging has become a component of our cytometric analysis, opening up whole new avenues for individual cell analysis. So where do we go from here? Laser technology is now available that makes our excitation capabilities essentially limitless. We will discuss developments in both laser and detector technology that should continue to increase both the number of parameters we can analyze, and expand the flexibility of our instruments to excite and detect the ever-expanding palette of fluorescent probes available to life scientists.