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Increased Mitochondrial Uptake of Rhodamine 123 During Lymphocyte Stimulation

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Increased Mitochondrial Uptake of Rhodamine 123 During Lymphocyte Stimulation Proc. Natl. Acad. Sci. USA Vol. 78, No. 4, pp. 2383-2387, April 1981 Cell Biology Increased mitochondrial uptake of rhodamine 123 during lymphocyte stimulation (flow cytometry/cycling-noncycling cells/supravital fluorescent probe) ZBIGNIEW DARZYNKIEWICZ, LISA STAIANO-COICO, AND MYRON R. MELAMED Memorial Sloan-Kettering Cancer Center, New York, New York 10021 Communicated by Lloyd J. Old, January 5, 1981 ABSTRACT The positively charged rhodamine analog rho- trifugation on Ficoll/Isopaque (Lymphoprep; Nyegaardo, Oslo, damine 123 accumulates specifically in the mitochondria of living Norway). The mononuclear cells were suspended in Eagle's cells. In the present work, the uptake of rhodamine 123 by indi- basal medium containing 15% fetal bovine serum and subcul- vidual lymphocytes undergoing blastogenic transformation in cul- tured in plastic dishes to remove most ofthe monocytes (3). The tures stimulated by phytohemagglutinin was measured by flow nonadhering cells were then suspended in Eagle's medium con- cytometry. A severalfold increase in cell ability to accumulate rho- taining penicillin, streptomycin, and 15% fetal bovine serum damine 123 was observed during lymphocyte stimulation. Maxi- and incubated at 37°C in the presence of PHA (phytohemag- mal dye uptake, seen on the third day ofcell stimulation, coincided glutinin M, GIBCO) as described (2, 3). The samples were with- in time with the peak of DNA synthesis (maximal number of cells drawn from these cultures at daily intervals. Other details ofthe in the S phase) and mitotic activity. A large intercellular variation are elsewhere 3). among stimulated lymphocytes, with some cells having fluores- cell culture system given (2, cence increased as much as 15 times in comparison with nonstim- Cell Staining. Acridine Orange. Cell staining with acridine ulated lymphocytes, was observed. Whereas the increased uptake orange (AO) was described in detail before (24). In this tech- ofrhodamine 123 also correlated with the increase in cellular RNA nique the cells are made permeable by Triton X-100 and then content, the correlation between the dye uptake and cell size exposed to 13 ,AM AO in the presence of NaEDTA and at an (measured by light scatter) was less apparent. As observed by UV AO-to-DNA phosphate molar ratio of >2 (4). The differential microscopy, the increased dye uptake during the blastogenesis was staining ofDNA and RNA is due to different modes ofdye bind- due, to a large extent, to an increase in number of mitochondria ing to those biopolymers. Namely, AO intercalates into double per cell. However, an additional increase in rhodamine 123 bind- helical DNA and in this form, as a monomer, fluoresces green ing per mitochondrion or per unit of mitochondrial membrane in (530 nm) in blue light (5). The dye interacting with single- stimulated cells could not be excluded. The present data indicate stranded RNA fluoresces metachromatically red with maximal that rhodamine 123 may be used as a supravital mitochondrial intensity at 640 nm (6). Because double-stranded RNA under- probe, discriminating between cycling and quiescent cells and hav- goes in situ denaturation in the presence of EDTA and AO, ing application in sorting functionally distinct cell subpopulations. nearly all cellular RNA is stained metachromatically (4). Stoi- chiometry of DNA and RNA staining by this method has been Johnson et al. (1) have recently reported that the fluorescent recently confirmed (7, 8). To evaluate the specificity ofstaining, laser dye rhodamine 123 directly and selectively stains mito- cells were treated with RNase A or DNase I (both obtained from chondria ofliving cells. These authors observed that the dye is Worthington) as described (2, 9). The data on RNA content taken up by mitochondria in a variety ofcell types without being (Table 1) refer to the RNase-sensitive portion of the red accumulated, even transiently, in other cell organelles (i.e., ly- fluorescence. sosomes, endocytic vesicles). Changes in mitochondrial orga- Rhodamine 123. Highly purified (laser dye quality) rhoda- nization, distribution, and shape induced by viral transforma- mine 123 was obtained from Eastman Organic Chemicals. The tion or colchicine treatment could be visualized easily after stock solution ofthe dye at 1 mg/ml was made in distilled water. staining with rhodamine 123 (1). The dye, reported not to be Further dye dilutions were made in Eagle's medium containing cytotoxic at concentrations up to 10 ,g/ml, thus may be used 15% fetal bovine serum and the dye was then added into cell supravitally as a mitochondrial probe. cultures to the final desired concentrations. In preliminary ex- We report here that the fluorescence of individual cells periments different concentrations (0.1-50 ,ug/ml) of the dye stained with rhodamine 123 can be measured by flow cytome- and different times of incubation (1-30 min) were tested with try, and we compare the rhodamine 123 binding ofnoncycling, respect to cell fluorescence and viability. Detailed results of quiescent lymphocytes with lymphocytes stimulated to prolif- these experiments will be presented elsewhere. In these stud- erate by phytohemagglutinin (PHA). In particular, the uptake ies, the optimal stainability of cells with rhodamine 123 was of rhodamine 123 is measured and correlated with progression obtained at 3-10 ,Ag/ml, and the plateau ofdye uptake was seen of stimulated lymphocytes through the cell cycle, as analyzed after 10 min ofincubation. Cell viability as assayed by the trypan by multiparameter flow cytometry based on simultaneous quan- blue or ethidium bromide exclusion tests was essentially un- titation of cellular RNA and DNA (2). changed during 24-hr culturing of rhodamine-labeled (10 ,g/ ml) cells. These conditions were used in the present studies and MATERIALS AND METHODS all the data presented on rhodamine 123 uptake were obtained Cells. Blood was collected by venipuncture of healthy vol- after preincubation ofcells with the dye at 10 ,g/ml for 10 min unteers and the leukocytes were separated by gradient cen- at 37°C. After the incubation, cells were centrifuged, resus- pended in Hanks' balanced salt solution, and examined by flow The publication costs ofthis article were defrayed in part by page charge cytometry. payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviations: PHA, phytohemagglutinin; AO, acridine orange. 2383 Downloaded by guest on September 28, 2021 2384 Cell Biology: Darzynkiewicz et al. Proc. Natl. Acad. Sci. USA 78 (1981) Stained cells from all cultures also were examined by UV concentrations and dye taken up by cells will, be given else- microscopy using a Leitz Orthoplan equipped with epifluores- where. The data on rhodamine 123 binding in this report were cent illumination and, in most observations, 485-nm excitation obtained after cell excitation at 488 nm and green fluorescence and 530-nm emission filters. To eliminate dead cells from the measurements at 515-575 nm (F&30). measured suspensions, the cells were incubated with a freshly Forward light scatter also was measured in samples stained prepared mixture of 0.25% trypsin and DNase I at 100 ,ug/ml with rhodamine; thus two parameters ofthose living cells were (both Worthington) at 370C for 20 min. Broken and dead cells, analyzed simultaneously (Fig. 2). Some of the data were recal- as well as isolated nuclei, are totally dissolved by these enzymes, culated for the mitogen-responsive subpopulations only (Table while living cells remain virtually unchanged (10). 1). The mitogen-responsive subpopulations were selected at a Measurements. Fluorescence (F) and light scatter of indi- given time point by using computer-interactive programs to vidual cells were measured in the FC 200Cytofluorograf(Ortho remove from the respective histograms cells with RNA, scatter, Diagnostic Instruments, Westwood, MA) interfaced to a Data or rhodamine fluorescence values characteristic ofthe nonstim- General minicomputer. Fluorescence pulses are generated in ulated cells. To this end, cells within the limits of ±3 SD ofthe this instrument by each cell as it passes in flow through a 488- respective means of RNA, scatter, or rhodamine fluorescence nm argon-ion laser beam. In the case of AO-stained cells, the of the nonstimulated cell populations ("0 time") were removed red (F>6w; measured in a band at 600-650 nm) and green (Fsw; from the histograms of, the PHA-stimulated lymphocytes and measured in a band at 515-575 nm) fluorescence emissions are the remaining cells were considered to represent the mitogen- separated optically and measured by separate photomultipliers, responsive subpopulation. Thus, at any given time point, this and the integrated values of the respective pulses for each cell subpopulation overlapped by less than 1% in fluorescence or are recorded in the computer. The width of the pulse also is scatter values with the quiescent lymphocytes. All data are measured and used to distinguish single cells from celldoublets, based on totals of5 X 103 cells measured per sample; cell dou- as described (11). blets and higher aggregates were excluded (11). The experiment The strongest emission of cells stained with rhodamine 123 was repeated thrice with essentially the same results. is green in blue light, as previously reported by Johnson et al. (1). A weaker red fluorescence also can be observed by UV microscopy after excitation at higher wavelengths (=530 nm) RESULTS AND DISCUSSION and using a longer wavelength (>600 nm) emission filter. The Peripheral blood lymphocytes are cells in a deep quiescent state detailed excitation and emission spectra of free dye at various (GQ, see ref. 10) having minimal RNA and 2C DNA content -I Ga -v%a-la .0 FIG. 1. Progression of lymphocytes through the cell cycle after their stimulation by PHA. The vertical dimension is the number of cells. Si- multaneous measurements ofcellular DNA (F530) and RNA (F>:0o) make it possible to analyze transition of cells from the quiescent compartment (G1Q) into the cycle (reflected as an increase in RNA) and their further progression through S and G2 + M (basedson F530 increase).
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