Biomaterials 35 (2014) 8748e8755
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
A phosphorescent iridium(III) solvent complex for multiplex assays of cell death Min Chen a, Yongquan Wu a, Yi Liu a, Huiran Yang b, Qiang Zhao b, Fuyou Li a, * a Department of Chemistry & Institutes of Biomedical Sciences & State Key Laboratory of Molecular Engineering of Polymers, Fudan University, 220 Handan Road, Shanghai 200433, PR China b Key Laboratory for Organic Electronics & Information Displays (KLOEID) and Institute of Advanced Materials (IAM), Nanjing University of Posts and Telecommnications, Nanjing 210046, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 May 2014 Accepted 22 June 2014 Available online 9 July 2014
Cell death involves loss of transport function and physical integrity of the plasma membrane, and plays a critical role in many human diseases. At present, the development of an effective visualization tool to monitor cell death remains a significant challenge. Here, a cyclometalated iridium(III) solvent complex [Ir(pdz)2(H2O)2]þ[OTf] (IrC1) was designed and synthesized as a phosphorescent indicator of cell death. IrC1 specifically stained the nuclei of dead cells over living cells rapidly (90%. Biocompatibility of IrC1 in other cell lines, such as HeLa and HepG2, was investigated with the MTT assay (Fig. S7). No significant loss of cell viability was observed at higher concentrations (up to 100 mM) of IrC1 following incubation for 24 h. The findings collectively indicate that IrC1 treatment causes no significant membranolytic effect, even at the highest concentration (90 mM), and presents low toxicity for luminescence cell imaging under the conditions applied (incubation time of ~10 min, concentration of 10 mM). Photostability characteristics of fluorescent materials present another consideration, particularly for study of the cell behavior process, since organic dyes undergo photobleaching in the longterm [13]. Here, photostability of IrC1 was compared with that of commercial organic dye under the same excitation conditions.
Fig. 1. Absorption (black line) and photoluminescence (red line) spectra of the IrC1 in DMSO/PBS (1:99, v/v), lex ¼ 405 nm. Inset: photographs showing the bright-field and luminescent emission of the IrC1 in DMSO/PBS (1:99, v/v) under excitation from UV lamp at 365 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Cell viability values (%) estimated by MTT proliferation test. Living KB cells were cultured in the presence of 0e90 mM at 37 C for 12 h and 24 h.
3. Results and discussion 3.1. Synthesis and photophysical properties of IrC1
M. Chen et al. / Biomaterials 35 (2014) 8748e8755
Relative to the organic probes, PI, the iridium complex, IrC1, exhibited higher photostability, as shown in Fig. 3. Specifically, continuous illumination in the focal plane induced significant photobleaching of PI, whereas IrC1 exhibited better photostability. To quantify the luminescence decay rates of IrC1 and PI, the luminescence intensity of each dye was plotted as a function of time. Moreover, the photostability of IrC1 was better than DAPI excited with a 405 nm laser (Fig. S8). Our findings clearly indicate that the bioimaging complex, IrC1, is suitable for continuous tracking studies over a long period of time. 3.3. Specific staining of nuclei of dead cells In view of its physiochemical properties and biocompatibility, we examined the applicability of IrC1 in cell imaging using confocal fluorescence microscopy. After incubation with 10 mM IrC1 for 10 min at 37 C, a very weak luminescence signal was detected in live cells (Fig. 4a), consistent with flow cytometry data (Fig. S9). This finding is significantly different from our previous data [19], although these are cyclometalated iridium(III) solvent complexes. The weak luminescence of living cells stained with IrC1 may be ascribed to two factors: either no IrC1 is taken up by the living cell or considerable amounts of the IrC1 complex enter living cells but only a small amount is converted to the luminescent form. To address this question, the amounts of iridium in IrC1-stained living cells were quantified via ICP-AEC (Fig. 4c). We detected ~1.6 fg Ir/ cell in IrC1-stained living cells, which is significantly lower than similar solvent iridium(III) complexes reported previously [19]. Accordingly, we conclude that weak cellular uptake of IrC1 occurs for living cells. Interestingly, when dead HeLa cells permeabilized with 4% paraformaldehyde were treated with IrC1, strong intracellular
Fig. 3. Comparison of photostability in laser scanning confocal microscopy (LSCM) imaging. The fixed KB cells were incubated with PI (green, lex ¼ 535 nm, lem ¼ 615 nm) and IrC1 (red, lex ¼ 405 nm, lem ¼ 625 nm), respectively. (a) Luminescent images of fixed cells incubated IrC1 complex; (b) Luminescent images of fixed cells incubated commercial dye PI; (c) Quantitative analysis of the changes in fluorescence intensities of IrC1 and PI. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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luminescence in nuclei was observed using confocal microscopy (Fig. 4b). Luminescence spectrum of IrC1 uptake in dead cells (Fig. S10) was similar to that of this complex in PBS, as shown in Fig. 1. Moreover, quantitation with line plots revealed large signal ratios (I2/I3 ~4000) between the nucleus (region 2) and cytoplasm (regions 1 and 3), as shown in Fig. 4d. To confirm the luminescence spots observed in the nucleus, a colocalization experiment with the commercial nuclear stain, DAPI, was performed using confocal fluorescence microscopy (Fig. 5). The luminescence signal (red) of IrC1 (excitation at 405 nm, emission at 625 ± 20 nm) was completely colocalized with that (blue) obtained with DAPI (excitation at 405 nm, emission at 450 ± 20 nm, as shown in Fig. 5d). Colocalization was evident from the bright pink spots in the nuclear region, suggesting that the nuclei of dead cells are targeted organelles of IrC1. Three-dimensional (3D) imaging of dead cells was additionally performed. Dead KB cells coloaded with IrC1 and the commercial membrane stain, DiI, were imaged by serially scanning at increasing depths along the z-axis. As shown in Fig. 6, the membrane was stained with DiI (blue, pseudo color), and the nuclear region clearly stained with IrC1 (red, pseudo color). Nuclei of KB cells were perfectly visualized in yz and xz cross-sectional images through 3D reconstruction of serial xy sections. Other types of dead cells, such as tumor HepG2 and HeLa, and normal HL-7702, were additionally subjected to nuclear staining (Fig. S11). Nuclearspecific staining in dead cells by IrC1 was evident, regardless of the cell type. This finding is similar to data obtained with the commercial nuclei-staining dye, PI, which conventionally distinguishes live over dead cells [24]. The results suggest that IrC1 can be effectively applied in a cell viability assay to distinguish live from dead cells and detect cell viability using flow cytometry. Using ICP-AES, the amounts of iridium element within IrC1treated dead cells were determined as 21.1 fg Ir/cell (Fig. 4c), which was significantly higher (>13-fold) than that in viable cells (1.6 fg Ir/ cell). These findings support the efficacy of IrC1 as a highly luminescent stain for distinguishing dead cells from viable cells. This performance in cell imaging is similar to that of a commercial nuclear stain, PI, leading to selective labeling of dead cells [24e26]. The reason underlying the selective staining of dead over living cells by IrC1 was further investigated. Lipophilicity of IrC1 was initially evaluated using the 1-octanol/water partition coefficient (logPo/w). The measured partition coefficient of IrC1 was 3.210 (in our previous report, the highest 1-octanol/water partition coefficient was 2.12 [19,27]), indicative of significant hydrophobic properties. A linear relationship between the IrC1 concentration and absorbance intensity was observed, as presented in Fig. S12, indicating that IrC1 is distributed evenly throughout the medium. High hydrophobicity may thus be responsible for IrC1 not permeating the membrane of living cells. The intact membrane of live cells excludes a variety of dyes, such as Trypan Blue [28] and PI [29], leading to no or minimal dye uptake by live cells and selective labeling of dead cells. To confirm this membrane resistance, liposomes composed of double phospholipid layers similar to cell membranes were used to encapsulate IrC1 to permeate membrane. After incubation of living cells with IrC1encapsuled liposomes for 10 h, IrC1 was located in the cytoplasm (Figs. S13 and S14), implying weak membrane penetration ability in living cells. In contrast, dead cells in which plasma and nuclear membranes became permeable were significantly stained by IrC1. Our findings indicate that membrane integrity is a key factor for IrC1-mediated discrimination of dead cells over viable cells. To determine changes in the luminescent behavior of IrC1 incubated with dead cells, the effects of specific biomolecules (including CT DNA and amino acids) on luminescent emission of IrC1 were examined. Upon addition of various residues (in
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Fig. 4. Confocal luminescent images of (a) living cells and (b) dead KB cells incubated with 10 mM IrC1 in PBS (pH 7.4) for 10 min at 37 C. (c) ICP-MS analysis of cellular uptake of IrC1. (d) Quantification of the luminescence intensity profile of IrC1-treated KB cells (lex ¼ 405 nm, lem ¼ 625 ± 20 nm).
particular, histidine), no significant changes in luminescent intensity were observed (Fig. S15), in contrast to our previous observation that histidine reacts with solvent iridium(III) complexes to cause luminescence enhancement [19]. Moreover, we
Fig. 5. Colocalization images of dead KB cells incubated with 10 mM IrC1 in PBS for 10 min at 25 C and then incubated with DAPI. (a) Blue channel for DAPI (lex ¼ 405 nm, lem ¼ 450 ± 20 nm); (b) Red channel for IrC1 (lex ¼ 405 nm, lem ¼ 625 ± 20 nm); (c) Brightfield image; (d) Colocalization of blue and red luminescence is shown as pink pixels. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 6. Three-dimensional luminescence images of fixed KB cells loaded with 10 mM IrC1 in DMSO/PBS (pH 7.4, 1:99, v/v) for 10 min at 37 C. The cell membrane was stained with DiI. Panel a is xy image obtained at z ¼ 0 mm, while panels b and c display the yz and xz cross sections (z ¼ 12to 11 mm) taken at the lines shown in panel a, respectively. The scale bar is 10 mm.
M. Chen et al. / Biomaterials 35 (2014) 8748e8755
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observed no obvious increase in the intensity of IrC1 after the addition of CT DNA (Fig. S16). This finding differs from data obtained with propidium iodide, which intercalates with DNA to markedly enhance dye fluorescence [24]. Further experiments are thus essential to establish the mechanism by which IrC1 selectively distinguishes dead cells over viable cells. The reason of IrC1 as an indicator in cell death is based on its different uptake performance between living and dead cells. The high hydrophobicity of this complex may be responsible for weak permeating the membrane of living cells, leading to minimal dye uptake. 3.4. Distinguishing apoptotic cells from live cells Apoptosis, the process of programmed cell death, is fundamentally important in numerous diseases, including cancer [30,31]. On the basis of the observation that specific staining of nuclei of dead cells is attributed to the physical integrity of the plasma membrane, it is assumed that IrC1 diffuses into apoptotic cells with altered plasma membrane [32e35]. To address this theory, we performed a colocalization experiment whereby apoptotic cells were incubated with IrC1 and a commercial apoptosis agent, FITClabeled Annexin V (stains early apoptotic and dead cells, lex ¼ 492 nm, lex ¼ 520 nm). Apoptotic cells detected upon introduction of H2O2 [36,37] (Fig. 7) were stained with both Annexin V-FITC and IrC1. Annexin V-FITC could not distinguish early apoptotic cells over dead cells (green channel). In contrast, IrC1 stained the nuclei of dead cells in a distinct manner to early apoptotic cells. Specifically, IrC1 selectively stained early apoptosis and dead cells at different locations in cell organisms. Moreover, the different intensities of intracellular IrC1 staining could be applied to distinguish early apoptotic from dead cells. IrC1 could not only discriminate between viable and dead cells, but also early apoptotic cells from live cells. To further confirm the application of IrC1 in distinguishing early apoptotic cells over dead cells, we used flow cytometry, which significantly enhances the accuracy of quantifying the appropriate cell populations. When live, early apoptotic or dead cells were
Fig. 8. Flow cytometric histogram profile of cellular uptake of IrC1 in KB cells. (a) KB cells were incubated with 10 mM IrC1 in DMSO/PBS (pH 7.4, 1:99, v/v) for 10 min in the culture mixed with live, early apoptosis, and dead cells; (b) Live, early apoptosis, or death KB cells were incubated with 10 mM IrC1 in DMSO/PBS (pH 7.4, 1:99, v/v) for 10 min. The x axes represent the fluorescent intensity profile from IrC1.
stained separately with IrC1, three peaks representing cells in the different conditions were observed, as shown in Fig. 8b. Furthermore, after staining of a mixture of live, apoptotic and dead cells for only 10 min by IrC1, three separate peaks appeared in one
Fig. 7. Confocal luminescence images of live (a), early apoptotic (b), and dead KB cells (c) incubated with Annexin V-FITC and then incubate with 10 mM IrC1 in PBS for 10 min at 37 C. Green channel for Annexin V-FITC(lex ¼ 488 nm, lem ¼ 530 ± 20 nm), red channel for IrC1(lex ¼ 405 nm, lem ¼ 625 ± 20 nm). The intensity profile is the quantification of the luminescence intensity in white line from the IrC1-treated KB cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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probe is rapid (
Contents lists available at ScienceDirect
Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
A phosphorescent iridium(III) solvent complex for multiplex assays of cell death Min Chen a, Yongquan Wu a, Yi Liu a, Huiran Yang b, Qiang Zhao b, Fuyou Li a, * a Department of Chemistry & Institutes of Biomedical Sciences & State Key Laboratory of Molecular Engineering of Polymers, Fudan University, 220 Handan Road, Shanghai 200433, PR China b Key Laboratory for Organic Electronics & Information Displays (KLOEID) and Institute of Advanced Materials (IAM), Nanjing University of Posts and Telecommnications, Nanjing 210046, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 May 2014 Accepted 22 June 2014 Available online 9 July 2014
Cell death involves loss of transport function and physical integrity of the plasma membrane, and plays a critical role in many human diseases. At present, the development of an effective visualization tool to monitor cell death remains a significant challenge. Here, a cyclometalated iridium(III) solvent complex [Ir(pdz)2(H2O)2]þ[OTf] (IrC1) was designed and synthesized as a phosphorescent indicator of cell death. IrC1 specifically stained the nuclei of dead cells over living cells rapidly (90%. Biocompatibility of IrC1 in other cell lines, such as HeLa and HepG2, was investigated with the MTT assay (Fig. S7). No significant loss of cell viability was observed at higher concentrations (up to 100 mM) of IrC1 following incubation for 24 h. The findings collectively indicate that IrC1 treatment causes no significant membranolytic effect, even at the highest concentration (90 mM), and presents low toxicity for luminescence cell imaging under the conditions applied (incubation time of ~10 min, concentration of 10 mM). Photostability characteristics of fluorescent materials present another consideration, particularly for study of the cell behavior process, since organic dyes undergo photobleaching in the longterm [13]. Here, photostability of IrC1 was compared with that of commercial organic dye under the same excitation conditions.
Fig. 1. Absorption (black line) and photoluminescence (red line) spectra of the IrC1 in DMSO/PBS (1:99, v/v), lex ¼ 405 nm. Inset: photographs showing the bright-field and luminescent emission of the IrC1 in DMSO/PBS (1:99, v/v) under excitation from UV lamp at 365 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Cell viability values (%) estimated by MTT proliferation test. Living KB cells were cultured in the presence of 0e90 mM at 37 C for 12 h and 24 h.
3. Results and discussion 3.1. Synthesis and photophysical properties of IrC1
M. Chen et al. / Biomaterials 35 (2014) 8748e8755
Relative to the organic probes, PI, the iridium complex, IrC1, exhibited higher photostability, as shown in Fig. 3. Specifically, continuous illumination in the focal plane induced significant photobleaching of PI, whereas IrC1 exhibited better photostability. To quantify the luminescence decay rates of IrC1 and PI, the luminescence intensity of each dye was plotted as a function of time. Moreover, the photostability of IrC1 was better than DAPI excited with a 405 nm laser (Fig. S8). Our findings clearly indicate that the bioimaging complex, IrC1, is suitable for continuous tracking studies over a long period of time. 3.3. Specific staining of nuclei of dead cells In view of its physiochemical properties and biocompatibility, we examined the applicability of IrC1 in cell imaging using confocal fluorescence microscopy. After incubation with 10 mM IrC1 for 10 min at 37 C, a very weak luminescence signal was detected in live cells (Fig. 4a), consistent with flow cytometry data (Fig. S9). This finding is significantly different from our previous data [19], although these are cyclometalated iridium(III) solvent complexes. The weak luminescence of living cells stained with IrC1 may be ascribed to two factors: either no IrC1 is taken up by the living cell or considerable amounts of the IrC1 complex enter living cells but only a small amount is converted to the luminescent form. To address this question, the amounts of iridium in IrC1-stained living cells were quantified via ICP-AEC (Fig. 4c). We detected ~1.6 fg Ir/ cell in IrC1-stained living cells, which is significantly lower than similar solvent iridium(III) complexes reported previously [19]. Accordingly, we conclude that weak cellular uptake of IrC1 occurs for living cells. Interestingly, when dead HeLa cells permeabilized with 4% paraformaldehyde were treated with IrC1, strong intracellular
Fig. 3. Comparison of photostability in laser scanning confocal microscopy (LSCM) imaging. The fixed KB cells were incubated with PI (green, lex ¼ 535 nm, lem ¼ 615 nm) and IrC1 (red, lex ¼ 405 nm, lem ¼ 625 nm), respectively. (a) Luminescent images of fixed cells incubated IrC1 complex; (b) Luminescent images of fixed cells incubated commercial dye PI; (c) Quantitative analysis of the changes in fluorescence intensities of IrC1 and PI. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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luminescence in nuclei was observed using confocal microscopy (Fig. 4b). Luminescence spectrum of IrC1 uptake in dead cells (Fig. S10) was similar to that of this complex in PBS, as shown in Fig. 1. Moreover, quantitation with line plots revealed large signal ratios (I2/I3 ~4000) between the nucleus (region 2) and cytoplasm (regions 1 and 3), as shown in Fig. 4d. To confirm the luminescence spots observed in the nucleus, a colocalization experiment with the commercial nuclear stain, DAPI, was performed using confocal fluorescence microscopy (Fig. 5). The luminescence signal (red) of IrC1 (excitation at 405 nm, emission at 625 ± 20 nm) was completely colocalized with that (blue) obtained with DAPI (excitation at 405 nm, emission at 450 ± 20 nm, as shown in Fig. 5d). Colocalization was evident from the bright pink spots in the nuclear region, suggesting that the nuclei of dead cells are targeted organelles of IrC1. Three-dimensional (3D) imaging of dead cells was additionally performed. Dead KB cells coloaded with IrC1 and the commercial membrane stain, DiI, were imaged by serially scanning at increasing depths along the z-axis. As shown in Fig. 6, the membrane was stained with DiI (blue, pseudo color), and the nuclear region clearly stained with IrC1 (red, pseudo color). Nuclei of KB cells were perfectly visualized in yz and xz cross-sectional images through 3D reconstruction of serial xy sections. Other types of dead cells, such as tumor HepG2 and HeLa, and normal HL-7702, were additionally subjected to nuclear staining (Fig. S11). Nuclearspecific staining in dead cells by IrC1 was evident, regardless of the cell type. This finding is similar to data obtained with the commercial nuclei-staining dye, PI, which conventionally distinguishes live over dead cells [24]. The results suggest that IrC1 can be effectively applied in a cell viability assay to distinguish live from dead cells and detect cell viability using flow cytometry. Using ICP-AES, the amounts of iridium element within IrC1treated dead cells were determined as 21.1 fg Ir/cell (Fig. 4c), which was significantly higher (>13-fold) than that in viable cells (1.6 fg Ir/ cell). These findings support the efficacy of IrC1 as a highly luminescent stain for distinguishing dead cells from viable cells. This performance in cell imaging is similar to that of a commercial nuclear stain, PI, leading to selective labeling of dead cells [24e26]. The reason underlying the selective staining of dead over living cells by IrC1 was further investigated. Lipophilicity of IrC1 was initially evaluated using the 1-octanol/water partition coefficient (logPo/w). The measured partition coefficient of IrC1 was 3.210 (in our previous report, the highest 1-octanol/water partition coefficient was 2.12 [19,27]), indicative of significant hydrophobic properties. A linear relationship between the IrC1 concentration and absorbance intensity was observed, as presented in Fig. S12, indicating that IrC1 is distributed evenly throughout the medium. High hydrophobicity may thus be responsible for IrC1 not permeating the membrane of living cells. The intact membrane of live cells excludes a variety of dyes, such as Trypan Blue [28] and PI [29], leading to no or minimal dye uptake by live cells and selective labeling of dead cells. To confirm this membrane resistance, liposomes composed of double phospholipid layers similar to cell membranes were used to encapsulate IrC1 to permeate membrane. After incubation of living cells with IrC1encapsuled liposomes for 10 h, IrC1 was located in the cytoplasm (Figs. S13 and S14), implying weak membrane penetration ability in living cells. In contrast, dead cells in which plasma and nuclear membranes became permeable were significantly stained by IrC1. Our findings indicate that membrane integrity is a key factor for IrC1-mediated discrimination of dead cells over viable cells. To determine changes in the luminescent behavior of IrC1 incubated with dead cells, the effects of specific biomolecules (including CT DNA and amino acids) on luminescent emission of IrC1 were examined. Upon addition of various residues (in
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Fig. 4. Confocal luminescent images of (a) living cells and (b) dead KB cells incubated with 10 mM IrC1 in PBS (pH 7.4) for 10 min at 37 C. (c) ICP-MS analysis of cellular uptake of IrC1. (d) Quantification of the luminescence intensity profile of IrC1-treated KB cells (lex ¼ 405 nm, lem ¼ 625 ± 20 nm).
particular, histidine), no significant changes in luminescent intensity were observed (Fig. S15), in contrast to our previous observation that histidine reacts with solvent iridium(III) complexes to cause luminescence enhancement [19]. Moreover, we
Fig. 5. Colocalization images of dead KB cells incubated with 10 mM IrC1 in PBS for 10 min at 25 C and then incubated with DAPI. (a) Blue channel for DAPI (lex ¼ 405 nm, lem ¼ 450 ± 20 nm); (b) Red channel for IrC1 (lex ¼ 405 nm, lem ¼ 625 ± 20 nm); (c) Brightfield image; (d) Colocalization of blue and red luminescence is shown as pink pixels. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 6. Three-dimensional luminescence images of fixed KB cells loaded with 10 mM IrC1 in DMSO/PBS (pH 7.4, 1:99, v/v) for 10 min at 37 C. The cell membrane was stained with DiI. Panel a is xy image obtained at z ¼ 0 mm, while panels b and c display the yz and xz cross sections (z ¼ 12to 11 mm) taken at the lines shown in panel a, respectively. The scale bar is 10 mm.
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observed no obvious increase in the intensity of IrC1 after the addition of CT DNA (Fig. S16). This finding differs from data obtained with propidium iodide, which intercalates with DNA to markedly enhance dye fluorescence [24]. Further experiments are thus essential to establish the mechanism by which IrC1 selectively distinguishes dead cells over viable cells. The reason of IrC1 as an indicator in cell death is based on its different uptake performance between living and dead cells. The high hydrophobicity of this complex may be responsible for weak permeating the membrane of living cells, leading to minimal dye uptake. 3.4. Distinguishing apoptotic cells from live cells Apoptosis, the process of programmed cell death, is fundamentally important in numerous diseases, including cancer [30,31]. On the basis of the observation that specific staining of nuclei of dead cells is attributed to the physical integrity of the plasma membrane, it is assumed that IrC1 diffuses into apoptotic cells with altered plasma membrane [32e35]. To address this theory, we performed a colocalization experiment whereby apoptotic cells were incubated with IrC1 and a commercial apoptosis agent, FITClabeled Annexin V (stains early apoptotic and dead cells, lex ¼ 492 nm, lex ¼ 520 nm). Apoptotic cells detected upon introduction of H2O2 [36,37] (Fig. 7) were stained with both Annexin V-FITC and IrC1. Annexin V-FITC could not distinguish early apoptotic cells over dead cells (green channel). In contrast, IrC1 stained the nuclei of dead cells in a distinct manner to early apoptotic cells. Specifically, IrC1 selectively stained early apoptosis and dead cells at different locations in cell organisms. Moreover, the different intensities of intracellular IrC1 staining could be applied to distinguish early apoptotic from dead cells. IrC1 could not only discriminate between viable and dead cells, but also early apoptotic cells from live cells. To further confirm the application of IrC1 in distinguishing early apoptotic cells over dead cells, we used flow cytometry, which significantly enhances the accuracy of quantifying the appropriate cell populations. When live, early apoptotic or dead cells were
Fig. 8. Flow cytometric histogram profile of cellular uptake of IrC1 in KB cells. (a) KB cells were incubated with 10 mM IrC1 in DMSO/PBS (pH 7.4, 1:99, v/v) for 10 min in the culture mixed with live, early apoptosis, and dead cells; (b) Live, early apoptosis, or death KB cells were incubated with 10 mM IrC1 in DMSO/PBS (pH 7.4, 1:99, v/v) for 10 min. The x axes represent the fluorescent intensity profile from IrC1.
stained separately with IrC1, three peaks representing cells in the different conditions were observed, as shown in Fig. 8b. Furthermore, after staining of a mixture of live, apoptotic and dead cells for only 10 min by IrC1, three separate peaks appeared in one
Fig. 7. Confocal luminescence images of live (a), early apoptotic (b), and dead KB cells (c) incubated with Annexin V-FITC and then incubate with 10 mM IrC1 in PBS for 10 min at 37 C. Green channel for Annexin V-FITC(lex ¼ 488 nm, lem ¼ 530 ± 20 nm), red channel for IrC1(lex ¼ 405 nm, lem ¼ 625 ± 20 nm). The intensity profile is the quantification of the luminescence intensity in white line from the IrC1-treated KB cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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probe is rapid (
