David Galbraith - Cell-type Discovery within Complex Tissues and Organs using Flow Cytometry and Sorting

  • Date: May 14, 2018
  • Time: 02:00 PM - 03:30 PM (Local Time Germany)
  • Speaker: David Galbraith
  • Location: Central Building
  • Room: Seminar Room
  • Host: Bernd Müller-Röber

The instruments developed for flow cytometry and sorting are designed for optimal operation using suspensions of cells having diameters in the range of 10-30 µm, simply because the greatest proportions of these instruments are deployed in the biomedical and clinical sectors. Many situations exist in which analysis is required of complex tissues and organs which are found as macroscopic three-dimensional elaborations of different cell types. My research, initially working with higher plants, has focused on ways to adapt, devise, and employ techniques of flow cytometry and sorting to questions arising within complex tissues and organs. We have employed two general approaches: (a) reduction of organs into suspensions of single cells (i.e. protoplasts), prior to flow analysis, and (b) production and flow analysis of homogenates prepared from these organs. This seminar will cover these aspects, including a description of methods to define different cell types through expression of Fluorescent Proteins, and to discover new sets of genes expressed within these cell types. I will demonstrate how these methods can be applied to the study of human disease. Finally, I will discuss the emerging field of agnostic discovery of cell types.

References:

  1. Galbraith, D.W., Harkins, K.R., Maddox, J.R., Ayres, N.M., Sharma, D.P., and Firoozabady, E. (1983). Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220:1049-1051.
  2. Sheen, J., Hwang, S., Niwa, Y., Kobayashi, H., and Galbraith, D.W. (1995). Green Fluorescent Protein as a new vital marker in plant cells. Plant Journal 8:777-784.
  3. Grebenok, R.J., Pierson, E.A., Lambert, G.M., Gong, F.-C., Afonso, C.L., Haldeman-Cahill, R., Carrington, J.C., and Galbraith, D.W. (1997). Green-Fluorescent Protein fusions for efficient characterization of nuclear localization signals. Plant Journal 11:573-586.
  4. Macas, J., Lambert, G.M., Dolezel, D., and Galbraith, D.W. (1998). NEST (Nuclear Expressed Sequence Tag) analysis: a novel means to study transcription through amplification of nuclear RNA. Cytometry 33:460-468.
  5. Birnbaum, K., Shasha, D.E., Wang, J.Y., Jung, J.W., Lambert, G.M., Galbraith, D.W., and Benfey, P.N. (2003). A gene expression map of the Arabidopsis root. Science 302:1956-1960.
  6. Barthelson, R.A., Lambert, G.M., Vanier, C., Lynch, R.M., and Galbraith, D.W. (2007). Comparison of the contributions of the nuclear and cytoplasmic compartments to global gene expression in human cells. B.M.C. Genomics 8:340.
  7. Zhang, C.Q., Barthelson, R.A., Lambert, G.M., and Galbraith, D.W. (2008). Characterization of cell-specific gene expression through fluorescence-activated sorting of nuclei. Plant Physiology 147:30-40.
  8. Grindberg, R.V., Yee-Greenbaum, J.L., McConnell, M.J., Novotny, M., O’ Shaughnessy, A.L., Lambert, G.M., Araúzo-Bravo, M.J., Lee, J., Fishman, M., Lin, X., Robbins, G.E., Lin, X., Venepally, P., Badger, J.H., Galbraith, D.W., Gage, F.H., and Lasken, R.S. (2013). RNA-Seq from single nuclei. Proceedings of the National Academy of Sciences U.S.A. 110:19802-19807.
  9. Samadder, P., Weng, N., Doetschman, T.C., Heimark, R., and Galbraith, D.W. (2016). Flow cytometry and single nucleus sorting for Cre-based analysis of changes in transcriptional states. Cytometry 89: 430-442.

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