© 2000 Wiley-Liss, Inc.
Cytometry 41:148 –149 (2000)
Letters to the Editor PROBING MITOCHONDRIAL PROBES The recent article by Keij et al. (1) criticizes the traditional distinction between mitochondrial mass probes (NAO, 10nonyl acridine orange; MFG, MitoFluor Green; MTG, MitoTracker Green) and mitochondrial potential probes (R123, rhodamine 123). They demonstrated that NAO, MFG, and MTG, often qualified as potential-insensitive probes, are not in fact reliable indicators of the mitochondrial mass. This is because their staining is significantly affected by potentialaltering drugs. Although we recognize the evidence of data reported and the relevance of the conclusions drawn as far as the non-neutrality of the so-called mass probes is concerned, we do not agree with the implicit validation of the use of potential-sensitive probes by flow cytometry. In the experimental model adopted by Keij et al. (1), cells are treated with drugs before incubation with probes. In this case, the total probe concentration, regardless of its distribution, well reflects the level of mitochondrial polarization. This situation, ideal for the purpose of the work, is rather different from that found in the majority of experiments, where changes in the mitochondrial potential are detected after the probe uptake. In this case, depolarization results in the release and spread of the probe in the cytosol with a net increase of fluorescence (2). In addition, the ATP depletion consequent to mitochondrial depolarization blocks all forms of ATPdriven efflux, thus permitting the cytoplasmic fluorescence to persist. Thus, in dynamic conditions, the sole datum of the fluorescence intensity, disregarding the intracellular distribution, is somewhat ambiguous. A second point that represents a potential error in the correlation of the fluorescence intensity to the mitochondrial potential is the intercellular variability of probe internalization. Our unpublished data indicate that the uptake of mitochondrial probes is extremely heterogeneous not only between different cell lines but also between cells of the same cell line, under the same experimental conditions, to the point that cells with intensely fluorescent mitochondria and cells with faintly stained mitochondria are often observed in the same microscopic field. This fact is due to multidrug resistance (MDR)-related behavior because it is abolished by treatment with MDR modulators. With regard to this property, staining with mitochondrial probes (R123, JC-1) represents a consolidated functional assay of MDR (3). With reference to the article in question, the assertion that “no localized fluorescence could be detected for 50 M FCCP, gramicidin and valinomycin” cannot easily be related to the reported paradoxical increase of MFG and MTG fluorescence after valinomycin (a classical K⫹ ionophore that dissipates the membrane potential). This unexpected finding should have received adequate morphological attention to prove or to exclude, for example,
mitochondrial swelling, which often accompanies depolarization. Swelling should correlate to an increase of fluorescence of potential-insensitive or less potential-sensitive probes. This might be a possible cause of the phenomenon observed. In conclusion, the article is meritworthy as it addresses for the first time the problem of the non-neutrality of the so-called mass probes. On the other hand, the limits of validity of the use of the other category of probes for the assessment of the mitochondrial potential by flow cytometry should also be taken into consideration. LITERATURE CITED 1. Keij JF, Bell-Prince C, Steinkamp JA. Staining of mitochondrial membranes with 10-nonyl acridine orange, MitoFluor Green and MitoTracker Green is affected by mitochondrial membrane potential altering drugs. Cytometry 2000;39:203–210. 2. Duchen MR, Leyssens A, Crompton M. Transient mitochondrial depolarizations reflect focal sarcoplasmic reticular calcium release in single rat cardiomyocytes. J Cell Biol 1998;142:975–988. 3. Ku ¨ hnel JM, Perrot JY, Faussat AM, Marie JP, Schwaller MA. Functional assay of multidrug resistant cells using JC-1, a carbocyanine fluorescent probe. Leukemia 1997;11:1147–1155.
Raffaella Isola Angela Maria Falchi Andrea Diana Giacomo Diaz* Department of Cytomorphology University of Cagliari Cittadella Universitaria Monserrato Italy ENRICHMENT FOR LATE-TELOPHASE CELL POPULATIONS USING FLOW CYTOMETRY In modern flow cytometry, “pulse processing” is a commonly used tool to exclude cell aggregates from cell-cycle analysis which involves deconvolution of single-parameter data with a three-compartment model (G1, S, G2 ⫹ M). However, in our view, exclusion of cell aggregates among which dividing cells of the final stage of mitosis, particularly late-telophase cells, are presumed to be present may affect mitotic indices evaluated by flow cytometry. In order to test this presumption, HeLa S3 (human cervical carcinoma) cells were stained with propidium iodide (PI) for DNA, and the area of the PI fluorescence signal in combination with the signal height (FL2-A vs. FL2-H) was utilized to discriminate late-telophase cells which might be present along with aggregates of two G1 cells prior to *Correspondence to: Giacomo Diaz, Department of Cytomorphology, University of Cagliari, Cittadella Universitaria, 09042 Monserrato, Italy. E-mail:
[email protected]
