The dye 10-N-nonyl-3,6-bis(dimethylamino)acridine (10-N-nonyl acridine orange) has been recently identified as a specific probe for cardiolipin (Ka = 2 x 10(6) M-1). It also interacts, at lower affinity (Ka = 7 x 10(4) M-1), with other acidic phospholipids [Petit, J. M., Maftah, A., Ratinaud, M. H. & Julien, R. (1992) Eur. J. Biochem. 209, 267-273]. In order to reduce the interference corresponding to monoacidic phospholipid binding, we have quantified cardiolipin by using a fluorimetric method based on the red fluorescence of the dye dimers formed at the diacidic phospholipid contact. Hence we have demonstrated that: (a) in yeast, the mitochondrion is the target of the dye whatever the cell metabolism; (b) membrane or protein organization and fatty acid unsaturation do not significantly modify the binding of 10-N-nonyl acridine orange. Using thin-walled vesicles, a linear relationship was established between the amount of cardiolipin and the red fluorescence emitted by the dye. Low red fluorescences were also observed with vesicles containing phosphatidylserine and phosphatidylinositol. However, at the same acidic phospholipid concentration, the fluorescence was much higher using cardiolipin-containing vesicles (fivefold that observed with phosphatidylserine-containing vesicles). Thus, 10-N-nonyl acridine orange was applied to cardiolipin quantification in yeast. This new method revealed that cells growing with a high glucose concentration contained 2.2 +/- 0.3 nmol cardiolipin/10(6) cells, whereas with lactate they contained about twice this amount (3.9 +/- 0.3 nmol cardiolipin).

Because flow cytometry permits the analysis of individual whole cells, one of the key requirements in selecting a probe is its ability to target the site of interest into cells. In addition, dyes must possess ideal properties (ie extinction coefficient, Stoke's shift) rendering them appropriate for this methodology. Other characteristics, such as fluorescence quenching and energy transfer, inherent to the staining, provide numerous applications in flow cytometry. The available fluorophores used in flow cytometry are classified according to their cellular incorporation and binding. Thus, probes are presented and discussed as follows: 1) dyes of cellular components (DNA, RNA, proteins, lipids); 2) probes of membrane potential; 3) fluorophores that are sensitive to their microenvironment (pH, calcium, etc); and 4) those used for measurement of enzymatic activities into cells.

The dye 10‐N‐nonyl‐3,6‐bis(dimethylamino)acridine (10‐N‐nonyl acridine orange) has been recently identified as a specific probe for cardiolipin (Ka= 2×106Mz−1). It also interacts, at lower affinity (Ka= 7×104M−1), with other acidic phospholipids [Petit, J. M., Maftah, A., Ratinaud, M. H. & Julien, R. (1992) Eur. J. Biochem. 209, 267–273]. In order to reduce the interference corresponding to monoacidic phospholipid binding, we have quantified cardiolipin by using a fluorimetric method based on the red fluorescence of the dye dimers formed at the diacidic phospholipid contact. Hence we have demonstrated that: (a) in yeast, the mitochondrion is the target of the dye whatever the cell metabolism; (b) membrane or protein organization and fatty acid unsaturation do not significantly modify the binding of 10‐N‐nonyl acridine orange. Using thin‐walled vesicles, a linear relationship was established between the amount of cardiolipin and the red fluorescence emitted by the dye. Low red fluorescences were also observed with vesicles containing phosphatidylserine and phosphatidylinositol. However, at the same acidic phospholipid concentration, the fluorescence was much higher using cardiolipin‐containing vesicles (fivefold that observed with phosphatidylserine‐containing vesicles). Thus, 10‐,N‐nonyl acridine orange was applied to cardiolipin quantification in yeast. This new method revealed that cells growing with a high glucose concentration contained 2.2±0.3 nmol cardiolipin/106cells, whereas with lactate they contained about twice this amount (3.9 ± 0.3 nmol cardiolipin).

No abstract

No abstract