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Staining of cellular mitochondria with LDS-751
Journal of Immunological Methods 257 Ž2001. 35–40 www.elsevier.comrlocaterjim
Staining of cellular mitochondria with LDS-751 D. Scott Snyder ) , Pamela L.C. Small Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 903 S. 4th St. Hamilton, MT 59840, USA Received 20 December 2000; received in revised form 1 March 2001; accepted 5 June 2001
Abstract We have found the dye LDS-751 to bind almost exclusively to mitochondria when incubated with viable, nucleated cells. Treatment of cells with the nuclear stain acridine orange and LDS-751 revealed little colocalization when the cells were examined by confocal microscopy. Staining with the dye rhodamine 123, which is known to bind polarized mitochondria, was virtually identical to the pattern observed with LDS-751. This staining pattern was observed to be consistent over a range of 0.02–20 mgrml LDS-751 and was consistent between both fibroblasts and monocytes. Depolarization of mitochondria with the mitochondrial depolarizing agents phenyl arsine oxide and carbonyl cyanide m-chlorophenylhydrazone ŽCCCP. dramatically reduced both LDS-751 staining, and rhodamine 123 fluorescence. Taken together, these results suggest that LDS-751 is excluded from the nucleus and binds the polarized membranes of mitochondria. Given this, interpretation of LDS-751 fluorescence as being indicative of nuclear status, as is commonly done to discriminate between leukocytes and erythrocytes, is unwarranted and may lead to erroneous conclusions if mitochondria become depolarized upon processing. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Mitochondria; LDS-751; Staining; Confocal microscopy
1. Introduction The dye LDS-751 has frequently been used in flow cytometry as a stain for DNA ŽFrey, 1995, 1997; Terstappen et al., 1988.. Given this condition, it is often assumed that the dye should stain the nuclei of intact cells. Unfortunately, no actual proof of this is available in the literature. Indeed, given the similarity of the dyes structure to that of styryl dyes ŽBereiter-Hahn, 1976., one might expect significant
) Corresponding author. Tel.: q1-406-363-9373X373; fax: q1406-363-9371. E-mail address: [email protected] ŽD.S. Snyder..
amounts of LDS-751 to localize to the mitochondria instead. From a technical perspective, this is important as LDS-751 is a commonly used probe in flow cytometry and has been used to differentiate between damaged and intact cells ŽTerstappen et al., 1988., follow apoptosis ŽFrey, 1995, 1997., and determine cell type ŽTerstappen et al., 1988.. The interpretation of results changes radically if LDS-751 binds to polarized mitochondrial membranes rather than DNA. In apoptosis, for example, there are both nuclear DNA changes such as chromatin condensation and fragmentation Žreviewed in Cohen, 1993., and changes in the polarization state of the mitochondria Žreviewed in Richter, 1993.. While DNA fragmentation
0022-1759r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 Ž 0 1 . 0 0 4 4 0 - 9
36
D.S. Snyder, P.L.C. Small r Journal of Immunological Methods 257 (2001) 35–40
is a hallmark of apoptosis, to conclude that cells are undergoing apoptosis because their mitochondria are not polarized may be erroneous. Likewise, as LDS-751 has a very long Stokes shift, use of this dye to stain mitochondria has certain advantages. Using a confocal fluorescence microscope, we show here that LDS-751 can be used to stain the mitochondria of living cells and is not taken up by the nucleus.
2. Materials and methods LDS-751 and rhodamine 123 were purchased from Molecular Probes ŽEugene OR.. 2.1. Cell culture L929 murine fibroblasts and RAW 264.7 monocytes were cultured at least 16 h in Dulbecco’s Modified Eagle’s Medium ŽDMEM. with 1% fetal bovine serum ŽFBS. on LabTek chambered coverglass wells at 37 8C with a humidified atmosphere containing 5% carbon dioxide prior to use.
witness consistently better staining patterns when it is diluted from ethanol. 2.3. Staining with other dyes Rhodamine 123 was diluted from stock solution in ethanol to a final concentration of 10 mM in HBSA, incubated 20 min at room temperature after which time the staining solution was decanted and replaced with buffer without dye. Acridine orange was added at a concentration of 5 mgrml, allowed to stain for 2–5 min, washed out with fresh buffer and imaged immediately. 2.4. Treatment with mitochondrial depolarizing agents For depolarization studies, cells were treated with dye as above, then rinsed with fresh buffer and treated with 7.5 mM digitonin and 100 mM phenylarsine oxide ŽPhAsO. for 30 min at 37 8C. Alternatively, cells were treated with 10 mM carbonyl cyanide m-chlorophenylhydrazone ŽCCCP. for 6 h at 37 8C prior to staining. 2.5. Confocal imaging
2.2. LDS-751 addition LDS-751 was made up as a stock solution at 0.2 mgrml in ethanol. It was diluted 100-fold in aqueous medium Žbuffer or growth medium. and 50 ml of diluted solution was added to coverslip wells containing L929 cells in 450 ml of either buffer or growth medium. Cultures were then incubated 20 min at room temperature after which time the medium was replaced with buffer containing no dye. The buffer used for these studies was 20 mM HEPES pH 7.4, 125 mM NaCl, 5 mM KCl, 0.62 mM MgSO4 , 1.8 mM CaCl 2 , 6 mM glucose ŽHBSA. and the growth medium was DMEM with FBS. No significant difference in staining pattern was observed between cells treated with LDS-751 diluted in buffer and cells treated in LDS-751 diluted in growth medium. For dose dependence studies, dilution directly from stock was performed so that the total concentration of ethanol never exceeded 1%. Note that although LDS-751 is also soluble in DMSO, we
Microscopy was done using a BioRad MRC 1024 laser scanning confocal imaging system connected to a Zeiss Axiovert 135 inverted microscope. The 488and 568-nm lasers were used either simultaneously or independently and 585-nm longpass filter was used to image LDS-751 with 568-nm excitation while green fluorescent dyes were imaged in a separate channel with a 522DF35 filter and 488 nm excitation. Magnification was set at 630 = , 2.3 zoom under an oil immersion lense. Laser power, gain and iris levels were manipulated with each dye independently so as to make sure that no image was produced in the channel not being visualized. Typical parameters were 3% laser power for both the 488and 568-nm lasers with iris 2.5 and gain of 800 in the 585EFLP channel but iris 4.0 and gain of 1350 in the 522DF35 channel. Alternatively, image subtraction should be considered with less fluorescent green channel dyes as LDS-751 is highly fluorescent and displays some crosstalk back to the green channel,
D.S. Snyder, P.L.C. Small r Journal of Immunological Methods 257 (2001) 35–40
37
Fig. 1. Murine L929 fibroblasts stained for 20 min with 2 mgrml LDS-751 and 10 mM rhodamine 123 then washed for 2–5 min with HBSA buffer and examined by confocal microscopy. ŽA. LDS-751 staining with 568-nm excitation and 585-nm longpass filter. ŽB. Rhodamine 123 staining with 488-nm excitation and 522DF35 emission filter. ŽC. Merge of images ŽA. and ŽB..
though it was not deemed necessary under the conditions used here. The system was controlled by the BioRad Lasersharp software package, which was also used to merge images and store data. Images can be merged in this system by selecting File, merge under the processing application, and selecting the appropriate files to merge under the color channel desired. Further processing was done using Adobe Photoshop 5.5 ŽAdobe Systems, San Jose CA.. Specifically, brightness and contrast were enhanced for printing. Typical adjustment was q20 for brightness and q30 for contrast.
is observed under 488-nm excitation but the pattern is identical Žthough much less intense. to that observed at 568 nm. LDS-751 concentration could be varied from 0.02 to 20 mgrml with little alteration in overall staining pattern. In no case was any significant staining of the nucleus observed. This was confirmed by sequential staining of the mitochondria
3. Results 3.1. Mitochondrial staining with LDS-751 When murine L929 fibroblasts are incubated with both LDS-751 and the mitochondrion specific probe rhodamine 123, virtually identical staining patterns are observed ŽFig. 1.. Both dyes produce a punctate staining pattern throughout the cytoplasm, characteristic of mitochondrial staining ŽJohnson et al., 1982.. Incubation with either dye alone produced similar staining patterns to those observed in coincubation Ždata not shown., indicating that the staining pattern of LDS-751 was not influenced by rhodamine 123 and vice versa. Note that some staining of LDS-751
Fig. 2. L929 fibroblasts stained 20 min with 2 mgrml LDS-751 Žred. and 5 min with 5 mgrml acridine orange Žblue..
38
D.S. Snyder, P.L.C. Small r Journal of Immunological Methods 257 (2001) 35–40
with LDS-751 then staining of the nucleus with acridine orange ŽFig. 2.. Nuclear staining by acridine orange Žblue. was observed to be largely exclusive of LDS-751 staining Žred.. 3.2. Treatment with digitonin, phenylarsine oxide and CCCP To determine whether LDS-751 staining of mitochondria is potential-dependent, we treated the cells with phenylarsine oxide ŽPhAsO., or CCCP, substances known to depolarize mitochondria ŽPastorino et al., 1996.. As PhAsO is not cell permeant, perme-
abilization of the cells with digitonin was required. Digitonin alone was not observed to significantly alter the staining pattern of the cells ŽFig. 3. but both CCCP and PhAsO dramatically reduced the staining levels of both LDS-751 ŽFig. 3.. In each case, LDS751 staining went from plainly visible to completely absent by visual inspection under identical examination parameters. Increase of laser power, gain and iris showed that mitochondria were indeed in the visual field but were only very weakly fluorescent. Staining of the cells with rhodamine 123 as a control showed an identical pattern, going from plainly visible to undetectable under identical examination pa-
Fig. 3. L929 fibroblasts stained with LDS-751 under control conditions ŽA., with digitonin ŽB., digitonin and PhAsO ŽC., or with CCCP. Images ŽA. and ŽB. are shown at q15 brightness and q25 contrast while images ŽC. and ŽD. are shown at q15 brightness and q45 contrast for clarity. To make images visible, gain was increased approximately 30% in ŽC. and laser power by 10-fold in ŽD..
D.S. Snyder, P.L.C. Small r Journal of Immunological Methods 257 (2001) 35–40
39
Fig. 4. RAW264.7 monocytes stained with rhodamine 123 ŽA, B. or LDS-751 ŽC, D.. Cells were incubated with growth medium ŽA, C. or growth medium and 10 mM CCCP ŽB, D.. To make the image visible, panel ŽD. was enhanced to q30 brightness, q45 contrast using Adobe Photoshop. Other panels were unaltered.
rameters, as expected since this dye is known to be potential-dependent Ždata not shown.. 3.3. Staining of monocyte mitochondria is potentialdependent Having shown that LDS-751 stains fibroblast mitochondria in a potential-dependent manner, we next investigated the staining patterns of the murine monocyte cell line RAW 264.7 upon exposure to LDS-751. A similar pattern of staining was witnessed to that of fibroblasts. Specifically, we ob-
served punctate staining of the cytoplasm and a lack of staining in the region of the cell where the nucleus might be expected to be ŽFig. 4.. Pretreatment of the cells with CCCP abrogated staining of both LDS-751 and the rhodamine 123 control.
4. Discussion The dye LDS-751 is known to increase its fluorescence upon binding to DNA. This has led to speculation that it may be useful as a nuclear local-
40
D.S. Snyder, P.L.C. Small r Journal of Immunological Methods 257 (2001) 35–40
ization probe ŽHaugland, 1996., and to its use in flow cytometry where it is often referred to as Aa vital nucleic acid stainB ŽTerstappen et al., 1988.. The pattern of staining we observe is not consistent with binding to nuclear, or even mitochondrial, DNA. The fact that LDS-751 colocalizes well with rhodamine 123 but only poorly with acridine orange points to staining of the mitochondria as opposed to the nucleus. Given that mitochondria have their own DNA, we originally thought that LDS-751 was binding to mitochondrial DNA. The fact that LDS-751 fluorescence is dramatically reduced when the cells are treated with the mitochondrial depolarizing agents PhAsO and CCCP, forced us to abandon this hypothesis. Instead, it seems much more plausible that LDS-751 stains polarized mitochondrial membranes, in a similar fashion to styryl dyes such as 2Ž4-Ždimethylamino .styryl.-N-methylpyridinium iodide ŽDASPMI, Bereiter-Hahn, 1976.. Indeed, examination of the chemical structure of LDS-751 shows certain similarities to this class of probes. In each case, a dimethylaminostyryl group is connected to a positively charged aromatic amine. The major differences are that LDS-751 has an extra double bond between the two aromatic moieties and has a larger aromatic amine group. Likewise, LDS-751 has a similar Stokes shift to DASPMI. Given both the structural and apparent functional similarities, it seems reasonable to classify LDS-751 as a styryl dye. The use of LDS-751 has a number of advantages in the staining of mitochondria. It is relatively fast, simple and usable on live cells with identical staining patterns being observed between monocytes and fibroblasts. Its large Stokes shift and long excitation wavelength make it compatible with green fluorescent probes. Sequential imaging of a 568-nm excitation image in the red channel followed by a 488-nm excitation image in the green channel can be used to minimize crosstalk between probes. The higher excitation wavelength of LDS-751 likewise gives this dye potential in the area of fluorescent resonance energy transfer when used in conjunction with green fluorescent dyes. By measuring the level of long wavelength fluorescence of LDS-751 upon excitation at a low wavelength and in the presence of a labeled molecule which absorbs at this low wavelength and
emits around 568, it should be possible to determine a relative distance of a target molecule to a polarized membrane. Given that membrane potential-dependent probes have been used in Escherichia coli ŽLopez-Amoros et al., 1997., it is even possible that the localization of target molecules in relation to the bacterial cell membrane could be determined by such a method. On the other hand, the increasingly prevalent use of this dye to discriminate cytometrically between erythrocytes and leukocytes in whole blood may need to be reevaluated. Although erythrocytes lack mitochondria, the fact that LDS-751 is potential-dependent means that leukocytes will only turn up positive if their mitochondrial membranes are polarized. Treatments that depolarize mitochondria in even a subset of leukocytes are likely to skew results in an unpredictable manner.
References Bereiter-Hahn, J., 1976. DASPMI as a fluorescent stain for mitochondria in situ. Biochim. Biophys. Acta 423, 1–14. Cohen, J.J., 1993. Apoptosis. Immunol. Today 14 Ž3., 126. Frey, T., 1995. Nucleic acid dyes for detection of apoptosis in live cells. Cytometry 21, 265. Frey, T., 1997. Correlated flow cytometric analysis of terminal events in apoptosis reveals the absence of some changes in some model systems. Cytometry 28, 253. Haugland, R.P., 1996. Handbook of Fluorescent Probes and Research Chemicals. 6th edn. Molecular Probes, Eugene, OR, p. 152. Johnson, L.V., Summerhayes, I.C., Chen, L.B., 1982. Decreased uptake and retention of rhodamine 123 by mitochondria in feline sarcoma virus-transformed mink cells. Cell 28, 7. Lopez-Amoros, R., Castel, S., Comas-Riu, J., Vives-Rego, J., 1997. Assessment of E. coli and Salmonella starvation by confocal laser microscopy and flow cytometry using rhodamine 123, DiBAC4Ž3., propidium iodide and CTC. Cytometry 29, 298. Pastorino, J.G., Simbula, G., Yamamoto, K., Glascott Jr., P.A., Rothman, R.J., Farber, J.L., 1996. The cytotoxicity of tumor necrosis factor depends on induction of the mitochondrial permeability transition. J. Biol. Chem. 271, 29792. Richter, C., 1993. Pro-oxidants and mitochondrial calcium: their relationship to apoptosis and oncogenesis. FEBS Lett. 325, 104. Terstappen, L.W., Meiners, H., Loken, M.R., 1988. A rapid sample preparation technique for flow cytometric analysis of immunofluorescence allowing absolute enumeration of cell subpopulations. J. Immunol. Methods 123, 103.
Staining of cellular mitochondria with LDS-751 D. Scott Snyder ) , Pamela L.C. Small Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 903 S. 4th St. Hamilton, MT 59840, USA Received 20 December 2000; received in revised form 1 March 2001; accepted 5 June 2001
Abstract We have found the dye LDS-751 to bind almost exclusively to mitochondria when incubated with viable, nucleated cells. Treatment of cells with the nuclear stain acridine orange and LDS-751 revealed little colocalization when the cells were examined by confocal microscopy. Staining with the dye rhodamine 123, which is known to bind polarized mitochondria, was virtually identical to the pattern observed with LDS-751. This staining pattern was observed to be consistent over a range of 0.02–20 mgrml LDS-751 and was consistent between both fibroblasts and monocytes. Depolarization of mitochondria with the mitochondrial depolarizing agents phenyl arsine oxide and carbonyl cyanide m-chlorophenylhydrazone ŽCCCP. dramatically reduced both LDS-751 staining, and rhodamine 123 fluorescence. Taken together, these results suggest that LDS-751 is excluded from the nucleus and binds the polarized membranes of mitochondria. Given this, interpretation of LDS-751 fluorescence as being indicative of nuclear status, as is commonly done to discriminate between leukocytes and erythrocytes, is unwarranted and may lead to erroneous conclusions if mitochondria become depolarized upon processing. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Mitochondria; LDS-751; Staining; Confocal microscopy
1. Introduction The dye LDS-751 has frequently been used in flow cytometry as a stain for DNA ŽFrey, 1995, 1997; Terstappen et al., 1988.. Given this condition, it is often assumed that the dye should stain the nuclei of intact cells. Unfortunately, no actual proof of this is available in the literature. Indeed, given the similarity of the dyes structure to that of styryl dyes ŽBereiter-Hahn, 1976., one might expect significant
) Corresponding author. Tel.: q1-406-363-9373X373; fax: q1406-363-9371. E-mail address: [email protected] ŽD.S. Snyder..
amounts of LDS-751 to localize to the mitochondria instead. From a technical perspective, this is important as LDS-751 is a commonly used probe in flow cytometry and has been used to differentiate between damaged and intact cells ŽTerstappen et al., 1988., follow apoptosis ŽFrey, 1995, 1997., and determine cell type ŽTerstappen et al., 1988.. The interpretation of results changes radically if LDS-751 binds to polarized mitochondrial membranes rather than DNA. In apoptosis, for example, there are both nuclear DNA changes such as chromatin condensation and fragmentation Žreviewed in Cohen, 1993., and changes in the polarization state of the mitochondria Žreviewed in Richter, 1993.. While DNA fragmentation
0022-1759r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 Ž 0 1 . 0 0 4 4 0 - 9
36
D.S. Snyder, P.L.C. Small r Journal of Immunological Methods 257 (2001) 35–40
is a hallmark of apoptosis, to conclude that cells are undergoing apoptosis because their mitochondria are not polarized may be erroneous. Likewise, as LDS-751 has a very long Stokes shift, use of this dye to stain mitochondria has certain advantages. Using a confocal fluorescence microscope, we show here that LDS-751 can be used to stain the mitochondria of living cells and is not taken up by the nucleus.
2. Materials and methods LDS-751 and rhodamine 123 were purchased from Molecular Probes ŽEugene OR.. 2.1. Cell culture L929 murine fibroblasts and RAW 264.7 monocytes were cultured at least 16 h in Dulbecco’s Modified Eagle’s Medium ŽDMEM. with 1% fetal bovine serum ŽFBS. on LabTek chambered coverglass wells at 37 8C with a humidified atmosphere containing 5% carbon dioxide prior to use.
witness consistently better staining patterns when it is diluted from ethanol. 2.3. Staining with other dyes Rhodamine 123 was diluted from stock solution in ethanol to a final concentration of 10 mM in HBSA, incubated 20 min at room temperature after which time the staining solution was decanted and replaced with buffer without dye. Acridine orange was added at a concentration of 5 mgrml, allowed to stain for 2–5 min, washed out with fresh buffer and imaged immediately. 2.4. Treatment with mitochondrial depolarizing agents For depolarization studies, cells were treated with dye as above, then rinsed with fresh buffer and treated with 7.5 mM digitonin and 100 mM phenylarsine oxide ŽPhAsO. for 30 min at 37 8C. Alternatively, cells were treated with 10 mM carbonyl cyanide m-chlorophenylhydrazone ŽCCCP. for 6 h at 37 8C prior to staining. 2.5. Confocal imaging
2.2. LDS-751 addition LDS-751 was made up as a stock solution at 0.2 mgrml in ethanol. It was diluted 100-fold in aqueous medium Žbuffer or growth medium. and 50 ml of diluted solution was added to coverslip wells containing L929 cells in 450 ml of either buffer or growth medium. Cultures were then incubated 20 min at room temperature after which time the medium was replaced with buffer containing no dye. The buffer used for these studies was 20 mM HEPES pH 7.4, 125 mM NaCl, 5 mM KCl, 0.62 mM MgSO4 , 1.8 mM CaCl 2 , 6 mM glucose ŽHBSA. and the growth medium was DMEM with FBS. No significant difference in staining pattern was observed between cells treated with LDS-751 diluted in buffer and cells treated in LDS-751 diluted in growth medium. For dose dependence studies, dilution directly from stock was performed so that the total concentration of ethanol never exceeded 1%. Note that although LDS-751 is also soluble in DMSO, we
Microscopy was done using a BioRad MRC 1024 laser scanning confocal imaging system connected to a Zeiss Axiovert 135 inverted microscope. The 488and 568-nm lasers were used either simultaneously or independently and 585-nm longpass filter was used to image LDS-751 with 568-nm excitation while green fluorescent dyes were imaged in a separate channel with a 522DF35 filter and 488 nm excitation. Magnification was set at 630 = , 2.3 zoom under an oil immersion lense. Laser power, gain and iris levels were manipulated with each dye independently so as to make sure that no image was produced in the channel not being visualized. Typical parameters were 3% laser power for both the 488and 568-nm lasers with iris 2.5 and gain of 800 in the 585EFLP channel but iris 4.0 and gain of 1350 in the 522DF35 channel. Alternatively, image subtraction should be considered with less fluorescent green channel dyes as LDS-751 is highly fluorescent and displays some crosstalk back to the green channel,
D.S. Snyder, P.L.C. Small r Journal of Immunological Methods 257 (2001) 35–40
37
Fig. 1. Murine L929 fibroblasts stained for 20 min with 2 mgrml LDS-751 and 10 mM rhodamine 123 then washed for 2–5 min with HBSA buffer and examined by confocal microscopy. ŽA. LDS-751 staining with 568-nm excitation and 585-nm longpass filter. ŽB. Rhodamine 123 staining with 488-nm excitation and 522DF35 emission filter. ŽC. Merge of images ŽA. and ŽB..
though it was not deemed necessary under the conditions used here. The system was controlled by the BioRad Lasersharp software package, which was also used to merge images and store data. Images can be merged in this system by selecting File, merge under the processing application, and selecting the appropriate files to merge under the color channel desired. Further processing was done using Adobe Photoshop 5.5 ŽAdobe Systems, San Jose CA.. Specifically, brightness and contrast were enhanced for printing. Typical adjustment was q20 for brightness and q30 for contrast.
is observed under 488-nm excitation but the pattern is identical Žthough much less intense. to that observed at 568 nm. LDS-751 concentration could be varied from 0.02 to 20 mgrml with little alteration in overall staining pattern. In no case was any significant staining of the nucleus observed. This was confirmed by sequential staining of the mitochondria
3. Results 3.1. Mitochondrial staining with LDS-751 When murine L929 fibroblasts are incubated with both LDS-751 and the mitochondrion specific probe rhodamine 123, virtually identical staining patterns are observed ŽFig. 1.. Both dyes produce a punctate staining pattern throughout the cytoplasm, characteristic of mitochondrial staining ŽJohnson et al., 1982.. Incubation with either dye alone produced similar staining patterns to those observed in coincubation Ždata not shown., indicating that the staining pattern of LDS-751 was not influenced by rhodamine 123 and vice versa. Note that some staining of LDS-751
Fig. 2. L929 fibroblasts stained 20 min with 2 mgrml LDS-751 Žred. and 5 min with 5 mgrml acridine orange Žblue..
38
D.S. Snyder, P.L.C. Small r Journal of Immunological Methods 257 (2001) 35–40
with LDS-751 then staining of the nucleus with acridine orange ŽFig. 2.. Nuclear staining by acridine orange Žblue. was observed to be largely exclusive of LDS-751 staining Žred.. 3.2. Treatment with digitonin, phenylarsine oxide and CCCP To determine whether LDS-751 staining of mitochondria is potential-dependent, we treated the cells with phenylarsine oxide ŽPhAsO., or CCCP, substances known to depolarize mitochondria ŽPastorino et al., 1996.. As PhAsO is not cell permeant, perme-
abilization of the cells with digitonin was required. Digitonin alone was not observed to significantly alter the staining pattern of the cells ŽFig. 3. but both CCCP and PhAsO dramatically reduced the staining levels of both LDS-751 ŽFig. 3.. In each case, LDS751 staining went from plainly visible to completely absent by visual inspection under identical examination parameters. Increase of laser power, gain and iris showed that mitochondria were indeed in the visual field but were only very weakly fluorescent. Staining of the cells with rhodamine 123 as a control showed an identical pattern, going from plainly visible to undetectable under identical examination pa-
Fig. 3. L929 fibroblasts stained with LDS-751 under control conditions ŽA., with digitonin ŽB., digitonin and PhAsO ŽC., or with CCCP. Images ŽA. and ŽB. are shown at q15 brightness and q25 contrast while images ŽC. and ŽD. are shown at q15 brightness and q45 contrast for clarity. To make images visible, gain was increased approximately 30% in ŽC. and laser power by 10-fold in ŽD..
D.S. Snyder, P.L.C. Small r Journal of Immunological Methods 257 (2001) 35–40
39
Fig. 4. RAW264.7 monocytes stained with rhodamine 123 ŽA, B. or LDS-751 ŽC, D.. Cells were incubated with growth medium ŽA, C. or growth medium and 10 mM CCCP ŽB, D.. To make the image visible, panel ŽD. was enhanced to q30 brightness, q45 contrast using Adobe Photoshop. Other panels were unaltered.
rameters, as expected since this dye is known to be potential-dependent Ždata not shown.. 3.3. Staining of monocyte mitochondria is potentialdependent Having shown that LDS-751 stains fibroblast mitochondria in a potential-dependent manner, we next investigated the staining patterns of the murine monocyte cell line RAW 264.7 upon exposure to LDS-751. A similar pattern of staining was witnessed to that of fibroblasts. Specifically, we ob-
served punctate staining of the cytoplasm and a lack of staining in the region of the cell where the nucleus might be expected to be ŽFig. 4.. Pretreatment of the cells with CCCP abrogated staining of both LDS-751 and the rhodamine 123 control.
4. Discussion The dye LDS-751 is known to increase its fluorescence upon binding to DNA. This has led to speculation that it may be useful as a nuclear local-
40
D.S. Snyder, P.L.C. Small r Journal of Immunological Methods 257 (2001) 35–40
ization probe ŽHaugland, 1996., and to its use in flow cytometry where it is often referred to as Aa vital nucleic acid stainB ŽTerstappen et al., 1988.. The pattern of staining we observe is not consistent with binding to nuclear, or even mitochondrial, DNA. The fact that LDS-751 colocalizes well with rhodamine 123 but only poorly with acridine orange points to staining of the mitochondria as opposed to the nucleus. Given that mitochondria have their own DNA, we originally thought that LDS-751 was binding to mitochondrial DNA. The fact that LDS-751 fluorescence is dramatically reduced when the cells are treated with the mitochondrial depolarizing agents PhAsO and CCCP, forced us to abandon this hypothesis. Instead, it seems much more plausible that LDS-751 stains polarized mitochondrial membranes, in a similar fashion to styryl dyes such as 2Ž4-Ždimethylamino .styryl.-N-methylpyridinium iodide ŽDASPMI, Bereiter-Hahn, 1976.. Indeed, examination of the chemical structure of LDS-751 shows certain similarities to this class of probes. In each case, a dimethylaminostyryl group is connected to a positively charged aromatic amine. The major differences are that LDS-751 has an extra double bond between the two aromatic moieties and has a larger aromatic amine group. Likewise, LDS-751 has a similar Stokes shift to DASPMI. Given both the structural and apparent functional similarities, it seems reasonable to classify LDS-751 as a styryl dye. The use of LDS-751 has a number of advantages in the staining of mitochondria. It is relatively fast, simple and usable on live cells with identical staining patterns being observed between monocytes and fibroblasts. Its large Stokes shift and long excitation wavelength make it compatible with green fluorescent probes. Sequential imaging of a 568-nm excitation image in the red channel followed by a 488-nm excitation image in the green channel can be used to minimize crosstalk between probes. The higher excitation wavelength of LDS-751 likewise gives this dye potential in the area of fluorescent resonance energy transfer when used in conjunction with green fluorescent dyes. By measuring the level of long wavelength fluorescence of LDS-751 upon excitation at a low wavelength and in the presence of a labeled molecule which absorbs at this low wavelength and
emits around 568, it should be possible to determine a relative distance of a target molecule to a polarized membrane. Given that membrane potential-dependent probes have been used in Escherichia coli ŽLopez-Amoros et al., 1997., it is even possible that the localization of target molecules in relation to the bacterial cell membrane could be determined by such a method. On the other hand, the increasingly prevalent use of this dye to discriminate cytometrically between erythrocytes and leukocytes in whole blood may need to be reevaluated. Although erythrocytes lack mitochondria, the fact that LDS-751 is potential-dependent means that leukocytes will only turn up positive if their mitochondrial membranes are polarized. Treatments that depolarize mitochondria in even a subset of leukocytes are likely to skew results in an unpredictable manner.
References Bereiter-Hahn, J., 1976. DASPMI as a fluorescent stain for mitochondria in situ. Biochim. Biophys. Acta 423, 1–14. Cohen, J.J., 1993. Apoptosis. Immunol. Today 14 Ž3., 126. Frey, T., 1995. Nucleic acid dyes for detection of apoptosis in live cells. Cytometry 21, 265. Frey, T., 1997. Correlated flow cytometric analysis of terminal events in apoptosis reveals the absence of some changes in some model systems. Cytometry 28, 253. Haugland, R.P., 1996. Handbook of Fluorescent Probes and Research Chemicals. 6th edn. Molecular Probes, Eugene, OR, p. 152. Johnson, L.V., Summerhayes, I.C., Chen, L.B., 1982. Decreased uptake and retention of rhodamine 123 by mitochondria in feline sarcoma virus-transformed mink cells. Cell 28, 7. Lopez-Amoros, R., Castel, S., Comas-Riu, J., Vives-Rego, J., 1997. Assessment of E. coli and Salmonella starvation by confocal laser microscopy and flow cytometry using rhodamine 123, DiBAC4Ž3., propidium iodide and CTC. Cytometry 29, 298. Pastorino, J.G., Simbula, G., Yamamoto, K., Glascott Jr., P.A., Rothman, R.J., Farber, J.L., 1996. The cytotoxicity of tumor necrosis factor depends on induction of the mitochondrial permeability transition. J. Biol. Chem. 271, 29792. Richter, C., 1993. Pro-oxidants and mitochondrial calcium: their relationship to apoptosis and oncogenesis. FEBS Lett. 325, 104. Terstappen, L.W., Meiners, H., Loken, M.R., 1988. A rapid sample preparation technique for flow cytometric analysis of immunofluorescence allowing absolute enumeration of cell subpopulations. J. Immunol. Methods 123, 103.