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Training in Flow cytometry practices for basic research with application in immunology and cancer
France (ANEMF) - UFR Sciences de santé Dijon, Dijon
Flow Cytometry Core Facility UMR1231 Inserm at the Faculty of Medicine 7 Bd Jeanne-d’ARC 21000 Dijon FRANCE
Dr. Anabelle Sequeira-Le Grand
Dr. Anabelle Sequeira-Le Grand
Type of Research Project
- Basic science
What is the background of the project?
Flow cytometry is a technology that has a wide range of application in medical sciences. It can be used as a complementary approach to understand immune pathologies, hematopoietic disorders and in a more general way, to monitoring the response to cancer therapy in patients. We have the purpose to strengthen the study of mitochondrial membrane potential, using the fluorescent probe JC-1. In fact, changes in membrane potential have long been known to represents early activation events. This approach represents good model to understand both, fluorescent probes and a functional application and demonstrates the use of the JC-1 probe for measuring mitochondrial membrane potential by flow cytometry. A valuable component of this measurement system is the possibility of making quantitative measurements changes in membrane potential. Regarding data analysis, we will provide a comprehensive training with the appropriate software specific for Flow Cytometry. This implies the understanding of spectral properties of fluorescent dyes in coherence with the instrumentation.
What is the aim of the project?
To provide the skills to propose Flox cytometry and its applications approaches in the medical field to open new ways for diagnosis and for therapy (using metabolic probes) and new alternatives for new therapeutic drugs.
What techniques and methods are used?
-Establish models of cell culture for the various applications: Immuno-marking; cellular cycle, study of mitochondrial membrane potential. -Study of the fluorescent process using lasers and optic filters. -Cell sorting, data analysis, and data management. Regarding the study of membrane potential, establish the experimental design: -quality of cells -establish a titration of the JC-1 probe -design the good controls (positive control, negative control) -find the best effector of depolarization for positive control -Data acquisition -Data analysis -Data interpretation and conclusions. -Study of the reproducibility of the test
What is the role of the student?
- The student will observe the practical experiments but will be highly involved in the analysis of the results
- If the project includes “lab work”
- the student will take active part in the practical aspect of the project
- The tasks of the student will be performed on his/her own
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
After a short training the student has to be able to help the core staff with their activities: analysis and cell sorting using flow cytometers, and in the same time learning about the different aspects of the day-to-day work for the research activity, gaining an overview of the conducted research. Learn about the dyes and controls used in flow experiments, the sensitivity of probes, the study of cells surface and intracellular antigens, the use of monoclonal antibodies and multicolor fluorescence.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
Yes, to complete the comprehension of the technology it is important to participate at least once a week in lectures or seminars provided by the tutor.
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- No specific outcome is expected
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
Basic Knowledge in biochemistry and immunology
Are there any legal limitations in the student’s involvement
The student can’t work alone in the lab and has to be informed about all risks concerning the work with biologic samples (human, mouse).
Type of students accepted
This project accepts: - Medical students - Graduated students (less than 6 months)
- M;Lu; J Levin; E. Sulpice; A. Sequeira-Le Grand; M Alemany; S. bellucci; JP Caen and ZC. Han 1999. The effect of AS2O3 on viability; proliferation and apoptosis in human megakaryocytic leukemia cell lines Exp. J. Hematol. 27: 845-852
- Desrumaux C; Lemaire-Ewing S; Ogier N; Yessoufou A; Hammann A; Sequeira-Le Grand A; Deckert V; Pais de Barros JP; Le Guern N; Guy J; Khan NA; Lagrost L. 2016 Plasma phospholipid transfer protein (PLTP) modulates adaptive immune functions through alternation of T helper cell polarization. Cell Mol Immunol. 2016 Nov;13(6):795-804. doi: 10.1038/cmi.2015.75. Epub 2015 Aug 31.
- Charles Thomas ; Antoine Jalil ; Charl_ene Magnani ; Minako Ishibashi Ronan Queré; Thibaut Bourgeois ; Victoria Bergas ; Louise Menegaut;; Danish Patoli ; Naig Le Guern ; Jerome Labbe ; Thomas Gautier ; Jean Paul Pais de Barros ; Laurent Lagrost ; David Masson LPCAT3 deficiency in hematopoietic cells alters cholesterol and phospholipid homeostasis and promotes atherosclerosis. 2018 Atherosclerosis 275 (2018) 409-418
- Margaux Sevin1;2; Lucia Kubovcakova3; Nicolas Pernet1;2; Sébastien Causse 1;2; Franck Vitte4; Jean Luc Villeval5;6; Catherine Lacout5;6; Marine Cordonnier1;2; Fernando Rodrigues-Lima7; Gaétan Chanteloup1;2; Matthieu Mosca5;6; Marie-Lorraine Chrétien8; Jean Noël Bastie1;2;8; Sylvain Audia8;; Paul Sagot8; Selim Ramla8; Laurent Martin8; Martin Gleave9; Valérie Mezger10;11;12; Radek Skoda3; Isabelle Plo5;6; 2018 HSP27 is a partner of JAK2-STAT5 and a potential therapeutic target in myelo fibrosis. NATURE COMMUNICATIONS |9:1431
- Andrea Cossarizza; Stefano Salvioli Current protocols in flow cytometry 01 May 2001. Flow Cytometric Analysis of Mitochondrial Membrane Potential Using JC‐1
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