Multiparametric analysis of cells with different mitochondrial membrane potential during apoptosis by polychromatic flow cytometry
| Autor: | Elisa Nemes, Andrea Cossarizza, Enrico Lugli, Erika Roat, Leonarda Troiano, Roberta Ferraresi, Marcello Pinti, Milena Nasi |
|---|---|
| Rok vydání: | 2007 |
| Předmět: |
Membrane Potential
Mitochondrial Membrane potential Programmed cell death Necrosis medicine.diagnostic_test Apoptosis U937 Cells Phosphatidylserine Transfection Biology Flow Cytometry General Biochemistry Genetics and Molecular Biology Flow cytometry Cell biology chemistry.chemical_compound chemistry medicine Humans Quercetin Propidium iodide medicine.symptom |
| Zdroj: | Nature Protocols. 2:2719-2727 |
| ISSN: | 1750-2799 1754-2189 |
| DOI: | 10.1038/nprot.2007.405 |
| Popis: | The analysis of changes in mitochondrial membrane potential (MMP) that can occur during apoptosis provides precious information on the mechanisms and pathways of cell death. For many years, the metachromatic fluorochrome JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide) was used for this purpose. Thanks to new dyes and to the technical improvements recently adopted in several flow cytometers, it is now possible to investigate, along with MMP, a variety of other parameters. Using three sources of excitation and polychromatic flow cytometry, we have developed a protocol that can be applied to cells undergoing apoptosis. In the model of U937 cells incubated with the chemopreventive agent quercetin (3,3',4',5,7-pentahydroxyflavone), we describe the detection at the single cell level of changes in MMP (by JC-1), early apoptosis (exposition of phosphatidylserine on the plasma membrane detected by annexin-V), late apoptosis and secondary necrosis (decreased DNA content by Hoechst 33342 and permeability of the plasma membrane to propidium iodide). The procedure can be completed in less than 2 h. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|