Open AccessFlow cytometric analysis of mitochondrial activity in situ: Application to acetylceramide‐induced mitochondrial swelling and apoptosis
Author(s) -
MacouillardPoulletier de Gannes Florence,
BelaudRotureau MarcAntoine,
Voisin Pierre,
Leducq Nathalie,
Belloc Francis,
Canioni Paul,
Diolez Philippe
Publication year - 1998
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/(sici)1097-0320(19981101)33:3<333::aid-cyto7>3.0.co;2-h
Subject(s) - mitochondrion , apoptosis , microbiology and biotechnology , biology , biophysics , flow cytometry , population , membrane potential , mitochondrial matrix , biochemistry , cytosol , medicine , environmental health , enzyme
Changes in mitochondrial matrix volume were studied both on isolated mitochondria and in situ on CHME 5 human microglia and monoblastoid U 937 cells using multiparametric flow cytometric analysis. The use of specific effectors of mitochondrial activity (oligomycin and KCN) allowed the demonstration, on whole cells, of a strict correlation between light scattering and mitochondrial volume changes: mitochondrial swelling induced a concomitant increase in forward scattering, and decrease in side scattering of the cell population. The technique was applied to the study of the early phases of acetyl‐ceramide‐induced apoptosis, which has been associated with mitochondrial dysfunction in several cellular systems. Acetyl‐ceramide caused a marked swelling of isolated rat liver mitochondria. Scatter modifications were also observed in both cell lines during the first hour of incubation with acetyl‐ceramide and were accompanied by an increase in DiOC 6 (3) fluorescence. The results imply that mitochondrial volume changes can be followed using flow cytometry and eventually used to assist in the interpretation of mitochondrial membrane potential variations obtained from fluorescence measurements. By applying this technique to 2 different cell lines, we demonstrated that mitochondrial swelling occurs during the early phases of acetyl‐ceramide treatment, but that the induction of apoptosis is cell type‐dependent. Cytometry 33:333–339, 1998. © 1998 Wiley‐Liss, Inc.