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FEB 21, 2018 6:00 AM PST

Multiparameter Cell Cycle Analysis

Speaker
  • Professor Emeritus (Oncology), Director Cytometry & Microscopy Core, Case Comprehensive Cancer Center, Co-Director Immune Function Core, Case Center for AIDS Research
    Biography
      James Jacobberger earned degrees in Microbiology and Immunology (BS, University of Nebraska (1977), MS, PhD, University of Rochester (1983)) and studied Experimental Pathology as a Post Doctoral Fellow (Dept. Pathology, University of Colorado (1983-1985). His PhD thesis and Post-Doctoral work emphasized concentrated efforts to apply cytometry to malaria and oncologenic virology problems. He was appointed Assistant Professor of Anatomy and Developmental Genetics (1985) at Case Western Reserve University and promoted to Associate Professor of General Medical Sciences (GMS-Oncology) with Tenure (1993), Professor of GMS-Oncology (2001), Professor Emeritus (2016). Dr. Jacobberger served as Associated Director for Shared Resources of the Case CCCC from 2006 - 2016. He created the current Case Comprehensive Cancer Center's Cytometry and Microscopy Shared Resource (CMSR) in 1985 with a single cell sorter. This (now) large facility thrives today with 4 Associate Directors, a staff of 3, and provides comprehensive cytometry and microscopy services to >100 laboratories per year. It is NIH funded as part of the Case Comprehensive Cancer Center and Case Center for AIDS Research. Dr. Jacobberger's research interests focus on cytometry with biological interests in cell cycle, oncogenesis, and biomarkers of clinical relevance in cancer. His research laboratory was continuously NIH funded for ~30 years. He has been an Associate Editor of the journal, Cytometry A. He has served on many NIH and other study sections, generally in the areas of instrumentation, biotechnology, and biomarkers. In April, 2018, Dr. Jacobberger will step down as Director of the CMSR but continue as part of the research services part of the CMSR. His future goals are to continue research and teaching.

    Abstract

    Most cell cycle assays amount to counting cells and/or measuring DNA content and deconvolving the resulting histogram into G1, S, G2+M, or G1, S, G2, and M.  Considerable effort has been put to correlating DNA content with RNA, protein, or specific epitopes to determine or discover additional compartments.  Here, we'll focus on flow cytometric analysis of DNA and specific epitopes.  In recent efforts, using DNA peak to mark anaphase/cytokinetic populations in a 6 parameter analysis (DNA-area, DNA-peak, Light Scatter, cyclin A2, cyclin B1, and phospho-S10-histone H3), we identify 15 compartments with more than 4 additional compartments representing apoptotic cells.

    Rational approaches to fixation and optimized staining protocols were worked out years ago.  We have examined many experimental variations on the basic protocols without uncovering any major improvements that result in higher quality data.  We are working currently on a “washless” staining assay, relying on a single high dilution to minimize background staining and relying on acoustic focusing to render the sample analyzable over a short period.  The results are striking. The S/N is equivalent to a fully washed sample; the minimized handling results in better cell recovery, and improved recovery results in better definition of cell cycle compartments with low cell numbers.  Additionally, the “washless” assay provides significant labor savings.  For research use only.  Not for use in diagnostic procedures.

    Learning Objectives:

    • overview of fixation & staining for intracellular epitopes
    • underlying logic and purpose of cell cycle analysis
    • in depth, analytical protocol for multiparameter analysis backbone

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