Dr. Lukas Flatz shares how the Biomark system contributed to new discoveries in his research
Fluidigm Technology Application
Fluidigm Technology Application
· Single cell gene expression
· BioMark system
· 96.96 integrated microfluidic dynamic chip
The study mentioned in this article is the work of Dr. Lukas Flatz when he was a postdoctoral fellow at the National Institutes of Health. He is currently a doctor and researcher in the dermatology department of the University of Lausanne, Switzerland.
Introduction
The National Center for Vaccine Research is part of the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (NIH), whose mission is to develop effective vaccines for human diseases. During his postdoctoral work at NIH, Dr. Lukas Flatz published an article on the intracellular response to AIDS vaccines at the Proceedings of the National Academy of Science s (Single-cell gene-expression profiling reveals qualitatively Distinct CD8 T cells elicited by different gene-based vaccines). He currently studies the role of T cells in skin diseases at the Department of Dermatology at the University of Lausanne.
challenge
CD8 T cells are known to provide immunoprotection against some pathogens following immunization. However, this protection depends on the heterogeneity and complexity of the cellular immune response caused by different vaccines.
Dr. Flatz began his research project using traditional gene expression analysis methods. He and his colleagues first injected 3 different vaccines into the mice. After 3 weeks, they extracted spleen cells from the mice, and treated the cells with P6 polypeptide at a rate, and stained the cytokines IFN-y, TNF-a and IL2 intracellularly. The results of these analytical methods demonstrate that CD8 T cells have similar cytokine functions under the action of the three vaccines. Next, they used microchip technology to further study these cells and began to discover some differences in cell response to different vaccines.
“We then extracted a question: How can we tell the difference between closely related immune responses?†he said. It was then that they found the technology of Fluidigm.
“It’s very interesting that when we applied Fluidigm’s Biomark technology to detect up to 96 different genes, we could really see significant differences,†he said.
solution
Through their experiments, the researchers identified subpopulations of CD8 T cells that were previously indistinguishable from single cells, demonstrating that they were induced by different vaccines.
Finally, the team found that subpopulations of T cells can be defined by gene expression in their central memory cells (CM) and effector memory cells (EM) based on their Eomes , Cxcr3 , Ccr7 , or Klrk1 , Klrg1 , and Ccr5 genes. .
As discussed in the discussion section: "In this study, analysis of gene transcription in a single cell can quantitatively resolve the response of CD8 T cells to different vaccines, which cannot be solved by conventional detection methods. This analysis is used in clinical practice. The relevance of protective immunity in these studies can be identified in pharmacodynamic experiments and in non-human primate studies."
“It’s very interesting that when we apply Fluidigm’s Biomark technology to detect up to 96 different genes, we can really see significant differences†- Dr. Lukas Flatz
For more information on FLUIDIGM single cell process technology, please visit
Technical advantages
Dr. Flatz said that single-cell gene expression analysis in dermatological studies can provide many technical advantages.
“When studying human diseases, you often can't get as many skin samples as you want. If you take too much tissue material, the patient will leave scars and cause harm. If you use traditional flow cytometry, you need a lot of materials, and Most of the time, only 15-20 parameters can be detected. With Biomark technology, you can detect up to 96 parameters in each cell, which provides many opportunities to simultaneously detect multiple gene transcription factors, protein expressions, etc. The method solves the problem of sample material limitation and allows us to obtain high resolution. We hope to find CD4 and CD8 cell populations associated with specific skin diseases such as psoriasis, and can also be applied to lupus erythematosus, eczema or other skin diseases. Common in immune response diseases. This can help us understand these diseases and possibly reclassify them into different subgroups, eventually identifying new therapeutic targets... we can get more information from one cell than from previous technologies. ."
resume
Dr. Lukas Flatz received his MD degree from the University of Berne, Switzerland, and has been involved in clinical and scientific research in immunology and dermatology. He is currently a researcher and doctor in the Department of Dermatology at the University of Lausanne, Switzerland. He previously served as a senior scientist at the Institute of Infectious Diseases at the University of Berne. He has worked as a postdoctoral fellow in the Virology Laboratory of the Institute of Vaccines of the National Institutes of Health, the Institute of Pathology and Immunology at the University of Geneva, Switzerland, and the Institute of Experimental Immunology at the University Hospital Zurich, Switzerland.
"With Biomark technology you can detect up to 96 parameters in every cell... we can get more information from one cell than with previous technology" - Dr. Flatz
Citation
Kerber R, T Rieger, C Busch., L Flatz, DD Pinschewer, BM Kümmerer, S Günther. 2011. Cross-species analysis of the replication complex of old world arenaviruses reveals two nucleoprotein sites involved in L protein function. J Virol. Dec 85 (23): 12518-28.
Flatz L, R. Roychoudhuri, M. Honda, A. Filali-Mouhim, JP. Goulet, N. Kettaf, M. Lin, M. Roederer, EK Haddad, RP Sekaly, GJ Nabel. 2011. Single cell gene-expression profiling Revises qualitatively distinct CD8 T cells elicited by different gene-based vaccines. Proc Natl Acad Sci USA, Apr 5; 108 (14): 5724-9.
Bergthaler A.*, L. Flatz*, AN Hegazy, S. Johnson, E. Horvath, M. Löhning, D. Pinschewer. 2010. Viral replicative capacity is the primary determinant of lymphocytic choriomeningitis virus persistence and immunosuppression. Proc Natl Acad Sci USA, Dec 14;107(50):21641-6. *contributed equally
Pinschewer DD, L.Flatz, R. Steinborn, E. Horvath, M. Fernandez, H. Lutz, M. Suter, A. Bergthaler. 2010. Innate and adaptive immune control of genetically engineered live-attenuated arenavirus vaccine prototypes. International Immunology , June 2010.
Flatz L., T. Rieger, D. Merkler, A. Bergthaler, T. Regen, M. Schedensack, L. Bestmann, A. Verschoor, M. Kreutzfeldt, W. Brück, U.-K. Hanisch, S. Günther , DD Pinschewer. 2010. T cell-dependence of Lassa fever pathogenesis. PLoS Pathogens , March 2010, Vol.6/3.
Flatz L., AN Hegazy, A. Bergthaler, A. Verschoor, C. Claus, M. Fernandez, L. Gattinoni, S. Johnson, F. Kreppel, S. Kochanek, M. van den Broek, A. Radbruch, F Levy, PH Lambert, CA Siegrist, NP Restifo, M. Loehning, A. Ochsenbein, GJ Nabel, DD Pinschewer. 2010. Development of replication-defective lymphocytic choriomeningitis virus vectors for induction of potent CD8+ T cell immunity. Nature Medicine , Feb . 17, 10.
Hegazy AN, M. Peine, C. Helmstetter, I. Panse, A. Frohlich, A. Bergthaler, L. Flatz, DD Pinschewer, A. Radbruch, M. Loehning,. 2010. Interferons direct Th2 cell reprogramming to generate a stable GATA-3+T-bet+ cell subset with combined Th2 and Th1 cell functions. Immunity , Jan. 29, 10.
Bergthaler, A., L. Flatz, A. Verschoor, AN Hegazy, M. Holdener, K. Fink, B. Eschli, D. Merkler, R. Sommerstein, E. Horvath, M. Fernandez, A. Fitsche, BM Senn , J. Sjef Verbeek, B. Odermatt, C.-A. Siegrist, DD Pinschewer. 2009. Impaired antibody response causes persistence of prototypic T cell-contained virus. PLoS Biology , Apr 7;7(4).
Lang, P., L. Cervantes-Barragan, A. Verschoor, AA Navarini, M. Recher, M. Pellegrini, L. Flatz, A. Bergthaler, K. Honda, B. Ludewig, PS Ohashi, KS Lang. 2009. Hematopoietic cell-derived interferon controls viral replication and virus-induced disease. Blood 113:1045-52.
Heikenwalder, M., M. Prinz, N. Zeller, KS Lang, T. Junt, S. Rossi, A. Tumanov, H. Schmidt, J. Priller, L. Flatz, T. Rulicke, AJ Macpherson, GA Hollander, SA Nedospasov, and A. Aguzzi. 2008. Overexpression of lymphotoxin in T cells induces fulminant thymic involution. Am J Pathol 172: 1555-1570.
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