Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry
| Autor: | Ana Cumano, Sophie Novault, Mariana Valente, Sandrine Schmutz |
|---|---|
| Přispěvatelé: | Cytométrie (Plate-forme), Institut Pasteur [Paris] (IP), Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto = University of Porto, Lymphopoïèse (Lymphopoïèse (UMR_1223 / U1223 / U-Pasteur_4)), Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), Instituto de Investigação e Inovação em Saúde (I3S), Unité de Recherche sur les Maladies Cardiovasculaires, du Métabolisme et de la Nutrition = Research Unit on Cardiovascular and Metabolic Diseases (ICAN), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Institut de Cardiométabolisme et Nutrition = Institute of Cardiometabolism and Nutrition [CHU Pitié Salpêtrière] (IHU ICAN), CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Schmutz, Sandrine, Institut Pasteur [Paris], Universidade do Porto, Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), Unité de Recherche sur les Maladies Cardiovasculaires, du Métabolisme et de la Nutrition = Institute of cardiometabolism and nutrition (ICAN), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [AP-HP], Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU) |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
embryonic heart [SDV]Life Sciences [q-bio] General Chemical Engineering Cell MESH: Flow Cytometry [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC] Cell Separation Animals Genetically Modified Mice Independent parameter MESH: Animals education.field_of_study medicine.diagnostic_test General Neuroscience MESH: Fluorescent Dyes Flow Cytometry Fluorescence Cell biology [SDV] Life Sciences [q-bio] Cellular Biology auto-fluorescence Issue 123 medicine.anatomical_structure MESH: Luminescent Proteins Biological system multi-parametric analysis Population Spectral flow [SDV.BC]Life Sciences [q-bio]/Cellular Biology Biology MESH: Cell Separation General Biochemistry Genetics and Molecular Biology Flow cytometry MESH: Animals Genetically Modified 03 medical and health sciences [SDV.BC.BC] Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC] medicine Animals education [SDV.BC] Life Sciences [q-bio]/Cellular Biology intestine MESH: Mice Fluorescent Dyes Cell specific General Immunology and Microbiology Luminescent Proteins 030104 developmental biology spectral Cytometry |
| Zdroj: | Journal of visualized experiments : JoVE Journal of visualized experiments : JoVE, 2017, 123, ⟨10.3791/55578⟩ Journal of visualized experiments : JoVE, JoVE, 2017, ⟨10.3791/55578⟩ |
| ISSN: | 1940-087X |
| DOI: | 10.3791/55578 |
| Popis: | International audience; Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management. |
| Databáze: | OpenAIRE |
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