Proc. Natl. Acad. Sci. USA Vol. 78, No. 4, pp. 2383-2387, April 1981 Cell Biology

Increased mitochondrial uptake of 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 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, 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- . 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- 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

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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). (a) Quiescent lymphocytes (G1Q) from the nonstimulated cultures are characterized by a Gi DNA content and by low RNA content. () Lymphocytes from the 24- hr-old PHA-treated cultures. About 50% ofcells with RNM above the G1Q level is observed. Few cells have entered S phase. (c) Two-day-old PHA cultures. About 80% of lymphocytes in these cultures have increased RNA content. Numerous cells are in S and G2 + M phases of the cycle. (d) Three-day-old PHA cultures. Stimulated cells have maximal RNA content and the highest proportion ofthem reside in S and G2 + M. (e) Four-day- old PHA culture. Lymphocytes on the way back to quiescence, losingRNA content and decreasing proliferation. A few dead cells present in these cultures have lowered F530 values but, with the exception of the nonstimulated population (a-), these cells are hidden by the G1 peaks and cannot be seen in this projection of the histograms. The population of dead cells disappears after incubation with DNase I and trypsin. Downloaded by guest on September 28, 2021 Cell Biology: Darzynkiewicz et al. Proc. Natl. Acad. Sci. USA 78 (1981) 2385 (see reviews 12 and 13). Upon stimulation by mitogens, lym- forward light scatter as presently measured relates mainly to cell phocytes asynchronously enter a longprereplicative phase char- size, although cell granularity and refractive index also influence acterized by a plethora of biochemical events occurring at the this measurement (14). Nonstimulated lymphocytes show rel- level of the cell membrane, cytoplasm, and nucleus. The most atively low and uniform values of the rhodamine 123 fluores- notable include changes in membrane permeability, a rise in cence and low values oflight scatter. Only minor changes in the the rate ofRNA synthesis and RNA content, increased synthesis fluorescence and light scatteroccur during the first 24 hrofstim- of proteins, and appearance of numerous enzymes associated ulation (Fig. 2b). On the second day, however, the distribution with DNA replication. Stimulated lymphocytes then enter the ofcells with respect to both fluorescence and light scatter values DNA replication phase and undergo mitosis; after three or four is clearly bimodal, as a subpopulation of PHA-responsive lym- rounds of cell division lymphocytes return to quiescence (12, phocytes with markedly increased fluorescence and light scatter 13). becomes apparent. The highest uptake ofrhodamine 123 is seen The kinetics of lymphocyte stimulation as reflected by in- on the third day; at that time the mean fluorescence of the re- crease in RNA content and cell progression through the cycle sponsive subpopulation is about 7 times higher than that of is illustrated in Fig. 1. During the first day after stimulation by either lymphocytes prior to stimulation (Fig. 2a) or of the non- PHA a subpopulation oflymphocytes with increased RNA con- responsive subpopulations in the same cultures. tent becomes apparent. Further rise in RNA content and an The responsive subpopulation is highly heterogeneous. increase in DNA as cells enter S and G2 + M phases is observed While some cells have only slightly increased rhodamine 123 on the second day, and on the third day of stimulation lym- fluorescence (10-20 units), there are also cells with fluorescence phocytes are characterized by maximal RNA content and the values above 100 units (Fig. 2d). The uptake of rhodamine 123 largest proportion ofcells in S and G2 + M phases. Finally, on is clearly diminished in 4-day-old PHA cultures. the fourth day, a decrease in RNA and lowered number ofS and Changes in light scatter properties of lymphocytes during G2 + M cells are seen. Even during maximal lymphocyte stim- stimulation have somewhat different kinetics than changes in ulation (third day) about 20% of cells in these cultures remain the uptake of rhodamine 123 (Fig. 2, Table 1). Namely, while with unchanged RNA and DNA content, representing the mi- no significant increase is observed during the first day, on the togen-nonresponsive subpopulations (GLQ). second day the PHA-responsive subpopulation had nearly 4- Cellular uptake ofrhodamine 123 and light scatter properties fold higher mean value oflight scatter as compared with control ofindivual cells were analyzed in the same cultures (Fig. 2). The or nonresponsive subpopulations. However, unlike rhodamine

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FIG. 2. Changes in lymphocyte light scatter properties and rhodamine 123 fluorescence after stimulation by PHA. Forward light scatter and green fluorescence (F530) due to the rhodamine 123 uptake were measured in lymphocyte populations from: nonstimulated cultures (a), and PHA- stimulated cultures after 24 hr (b), 2 days (c), 3 days (d), or 4 days (e). The few dead cells present in these cultures did not show any measurable fluorescence and had light scatter values below the triggering threshold in these measurements; these cells are absent in the histograms. Downloaded by guest on September 28, 2021 2386 Cell Biology: Darzynkiewicz et al. Proc. Natl. Acad. Sci. USA 78 (1981) Table 1. Changes in cellular RNA, light scatter, and cell mitochondria; at that time the cytoplasm and nucleus exhibit ability to accumulate rhodamine 123 during lymphocyte a diffuse, homogeneous fluorescence. stimulation The present studies indicate that during cell transition from quiescence to the cycle, a severalfold increase in rhodamine 123 Time after Arbitrary units uptake takes place. This increase reflects to a large extent the PHA, days RNA content Light scatter Rhodamine uptake increase in number and in total mass of mitochondria per cell. 0 7.4 (1.0) 10.6 (1.0) 7.0 (1.0) An increase in number of mitochondria during lymphocyte 1 20.6 (2.8) 14.7 (1.4) 16.1 (2.3) stimulation has been reported by Inman and Cooper (15). The 2 33.5 (4.5) 33.2 (3.1) 22.0 (3.1) present data, however, cannot exclude a possible increase in 3 46.1 (6.2) 31.8 (3.0) 51.1 (7.3) rhodamine 123 binding per mitochondrion or per unit of mi- 4 25.5 (3.5) 36.6 (3.5) 24.9 (3.6) tochondrial membrane during cell activation. In these rounded Cell samples were withdrawn from the same lymphocyte cultures cells the total number of mitochondria cannot be counted ac- incubated with PHA for up to 4 days. Cells were then analyzed for RNA curately, nor can the planimetric measurements of the mito- after staining with AO, as described in the legend to Fig. 1, or for light chondrial surface be done. scatter and rhodamine 123 uptake (see Fig. 2). The arbitrary units are Molecular mechanisms related to the specificity of binding equivalent to channel number. The mean values of all mononuclear cells (as shown in the histograms, Figs. la and 2a-i.e., prior to ad- ofrhodamine 123 by mitochondria are unknown. Johnson et al. dition of PHA) are given for the 0 time point. Other figures represent (1) have suggested that the high electronegativity of the intact mean values of the mitogen-responsive subpopulations on days 1, 2, mitochondrial membrane may be responsible for the specific 3, and 4 after PHA. The mitogen-responsive subpopulations were se- dye binding. Indeed, at physiological pH, only the positively lected by using a computer-interactive program to remove from the charged rhodamine analogs show an affinity towards mitochon- respective histograms cells with the RNA, scatter, or rhodamine flu- dria; neutral or negatively charged analogs do not stain these orescence values characteristic ofthe nonstimulated lymphocytes. The figures in the RNA content column represent the RNase-sensitive por- organelles (1). It is possible, therefore, that the intensity of tion of the F>600 fluorescence after AO staining. All figures in paren- mitochondrial staining may correlate with the activity of the theses show the increase of the measured parameters with respect to oxidoreductive complexes responsible for the maintenance of the nonstimulated cells (0 time = 1.0). the electronegativity ofthe mitochondrial membrane, and thus the rhodamine 123 may be a direct marker of the energy-sup- 123 uptake, mean values of light scatter of the responsive sub- plying metabolic processes. The maximal uptake of rhodamine population did not change further during the subsequent third 123 on the third day of stimulation, as presently observed, co- and fourth day of culturing. During maximal stimulation (third incides in time with: (i) maximal number ofcells in the S phase day) about 20% of the lymphocytes remained with unchanged and (ii) maximal RNA content of the S cells (see Figs. 1 and 3). values of the rhodamine 123 uptake and light scatter-i.e., a Because the rate of DNA replication, per cell, in stimulated similar proportion to those not responding to PHA by the RNA lymphocytes is highly correlated with RNA content (16) the increase (Fig. 1). present data suggest that, in turn, a correlation may exist be- Cell morphology and viability were analyzed by microscopy tween the rate of DNA synthesis and rhodamine 123 uptake. in all cultures and were compared with cellular ability to ac- Thus, the increased dye uptake may be a reflection of the in- cumulate rhodamine 123. By the standard trypan blue exclusion creased energy requirements of the rapidly dividing cells. test there were less than 1% dead cells prior to stimulation and Regardless ofthe mechanism of rhodamine 123 binding, the 8%, 12%, 16%, and 18% dead cells on day 1, 2, 3, and 4 after present data indicate that the dye taken up specifically by mi- stimulation, respectively. When the samples were preincu- tochondria of living cells may be used as a supravital stain dis- bated simultaneously with trypan blue and rhodamine and then criminating between cycling and quiescent cells. This fluoro- analyzed by combined light and UV microscopy, the cells ex- chrome, therefore, may be used for supravital cell staining and cluding trypan blue exhibited bright green fluorescence of cy- sorting of functionally distinct subpopulations for their further toplasmic structures typical of mitochondrial shapes as de- analysis-i.e., by precursor incorporation studies or cloning. scribed by Johnson et al. (1), while nearly all dead cells were not fluorescent. Most of the fluorescent structures had either oval or rod-like appearance, and were more numerous in blast cells from 3- to 4-day cultures than in small lymphocytes. Nuclei of those cells did not show any fluorescence. There was, how- :t40 r. ever, originally a small number ofcells (1-2%) characterized by .0 ac bright and homogenous fluorescence of nuclei and cytoplasm. n: aE ~a 04) Nearly all ofthem showed cytoplasmic budding typical ofdying CZ

cells. Their number increased with the time of observation, -62 a. a~ especially within the UV-illuminated field. Such cells, in time, 0 Q become stainable with trypan blue and then lose their a2 fluorescence. 0 Prior to measurements or observations by microscopy, some cell samples were also preincubated with DNase I at 100 pkg/ ml and 0.25% trypsin, at 370C for 20 min. In such preparations 100% ofcells exclude trypan blue because all the dead cells and isolated nuclei are lysed by a mixture ofthese enzymes. All cells Time, days in these preparations had typical mitochondrial fluorescence staining patterns with negative nuclei. Thus, we have been able FIG. 3. Changes- in rhodamine 123 uptake during lymphocyte stimulation as compared with cell distribution at different phases of to confirm the observations ofJohnson et al. (1) that rhodamine the cell cycle. The proportions of cells at different phases of the cycle 123 selectively stains mitochondria of living cells and does not are plotted against the time of culturing with PHA. Broken line (Rho) stain cells that are dead. During cell death, however, there is shows changes in mean values of green fluorescence of PHA-stimu- a transient phase when the dye is apparently released from lated lymphocytes (GjQ cells excluded) incubated with rhodamine 123. Downloaded by guest on September 28, 2021 Cell Biology: Darzynkiewicz et al. Proc. Natl. Acad. Sci. USA 78 (1981) 2387

The possible general usefulness of this probe in discriminating 7. Coulson, P., Bishop, A. 0. & Lenarduzzi, R. (1977) J. Histo- quiescent versus cycling cell subpopulations should be further chem. Cytochem. 25, 1147-1153. evaluated in other cell systems. 8. Bauer, K. D. & Dethlefsen, L. A. (1980) J. Histochem. Cyto- chem. 28, 493498. 9. Traganos, F., Darzynkiewicz, Z., Sharpless, T. & Melamed, M. We thank Miss Robin Nager for her help in preparation ofthe manu- R. (1977) J. Histochem. Cytochem. 25, 46-56. script. The work was supported by U.S. Public Health Service Grants 10. Darzynkiewicz, Z., Traganos, F. & Melamed, M. R. (1980) Cy- 1-ROI-CA23296, CA-28704, and 1-26-CA14134 through the National tometry 1, 98-108. Bladder Cancer Project. 11. Sharpless, T., Traganos, F., Darzynkiewicz, Z. & Melamed, M. R. (1975) Acta Cytol. 19, 577-581. 1. Johnson, L. V., Walsh, M. L. & Chen, L. B. (1980) Proc. Natl. 12. Ling, N. R. & Kay, J. E. (1975) Lymphocyte Stimulation (North- Acad. Sci. USA 77, 990-994. Holland, Amsterdam). 2. Darzynkiewicz, Z., Traganos, F., Sharpless, T. & Melamed, M. 13. Loeb, L. A. (1975) in Developments in Lymphoid Cell Biology, R. (1976) Proc. Natl. Acad. Sci. USA 73, 2881-2884. ed. Gottlieb, A. A. (CRC, Cleveland, OH), pp. 103-132. 3. Darzynkiewicz, Z., Evenson, D. P., Staiano-Coico, L., Sharp- 14. Salzman, G. C., Mullaney, P. F. & Price, B. J. (1979) in Flow less, T. & Melamed, M. R. (1979)J. Cell Physiol. 100, 425-438. Cytometry and Sorting, eds. Melamed, M. R., Mullaney, P. F. 4. Darzynkiewicz, Z., Traganos, F., Sharpless, T. & Melamed, M. & Mendelsohn, M. L. (Wiley, New York), pp. 105-135. R. (1975) Exp. Cell Res. 95, 143-153. 15. Inman, D. R. & Cooper, E. H. (1963)J. Cell Biol. 19, 441-445. 5. Lerman, L. S. (1963) Proc. Natl. Acad. Sci. USA 49, 94-102. 16. Darzynkiewicz, Z., Evenson, D., Staiano-Coico, L., Sharpless, 6. Bradley, D. F. & Wolf, M. F. (1959) Proc. Natl. Acad. Sci. USA T. & Melamed, M. R. (1979) Proc. Natl. Acad. Sci. USA 76, 43, 944-952. 358-362. Downloaded by guest on September 28, 2021