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A cell-penetrating ratiometric probe for simultaneous measurement of lysosomal and cytosolic pH change
Talanta 178 (2018) 355–361
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A cell-penetrating ratiometric probe for simultaneous measurement of lysosomal and cytosolic pH change
MARK
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Meng-Chan Xia, Lesi Cai, Sichun Zhang , Xinrong Zhang Department of Chemistry, Beijing Key Laboratory of Microanalytical Methods and Instrumentation, Tsinghua University, Beijing 100084, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords: Intracellular pH Fluorescent probes Sensors Cell imaging Cell-penetrating peptides
A new ratiometric fluorescent probe based on cell-penetrating peptides (CPPs) was constructed for whole-cell pH mapping and simultaneous measurement of pH changes in the cytoplasm and lysosomes. The arginine-rich CPP, R12K worked as linker, carrier and part of the fluorophore. Benefiting from R12K, the fluorescent probe is completely water soluble, membrane permeable and well biocompatible. It shows high selectivity, sensitivity and reversibility to pH fluctuations. The ratio of fluorescence intensities F519/F582 increased from 0.2 to 9.2 over the pH range from 3.3 to 8.1. Intracellular pH mapping was successfully realized owing to the wide distribution of the probe in live cells (even in nucleus). Moreover, cytosolic and lysosomal pH change caused by the stimuli can be simultaneously detected. Compared to other ratiometric pH probes, RhB-R12K-FITC can provide more precise information about H+ redistribution between different cellular compartments.
1. Introduction Cells are highly compartmentalized and organized. Different cellular compartments with varied pH values provide distinct conditions for optimal operation [1]. The lumens of lysosomes and endosomes are faintly acidic [2,3], whereas nucleus and cytoplasm are near neutral [4,5]. The pH changes of lysosomes and cytoplasm are closely related in basic cellular activities, such as metabolism [1], oxidative stress [6] and apoptosis [7,8]. Abnormal pH values can lead to cellular dysfunction and indicate the risk of cancer [9–11], neurodegenerative disorders [12,13] and cardiovascular disease [14]. Monitoring intracellular pH homeostasis and how pH is regulated can improve our understanding of physiological and pathological processes. Fluorescent imaging has occupied a decisive position in live cell imaging attributed to their high selectivity, excellent sensitivity and spatiotemporal resolution [15–18]. Ratiometric fluorescent probes give more accurate analysis on intracellular pH, because of the corrections of some systematic errors resulted from dye leakage, photobleaching and optical path length. Although various small-molecule probes and nanoprobes were developed for ratiometric pH sensing [19–27], most of them focus on one specific organelle. For in-depth research on H+ redistribution in physiological process and cellular behavior, simultaneous pH analysis of different cellular compartments is urgently required. Using a combination of multiple organelle-specific pH sensors is much more complicated [28] and researchers are devoted to developing effective pH indicators for intracellular pH mapping [19,21,29]. As far
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as we know, few pH probes allow simultaneous detection of cytosolic and lysosomal pH change. Cell penetrating peptides (CPPs) have been promising carriers to facilitate cellular internalization [30,31]. Our group previously reported a spirolactam derivative RhB-R12K by conjugating the fluorescent dye rhodamine B to CPP [32]. Here, we fully took advantage of the remaining active groups in the CPP and constructed a ratiometric fluorescent pH probe RhB-R12K-FITC. The new probe, which shows high selectivity and sensitivity to H+, possesses excellent membrane permeability, water-solubility and low toxicity. It can even be phagocytized by nucleus due to the typical feature of arginine-rich CPPs [33]. Its remarkable properties and wide distribution in live cells enable relatively successful intracellular pH mapping. The pH value of specific compartment can be measured individually. Furthermore, the pH fluctuations and H+ redistribution between cytoplasm and lysosomes caused by oxidative stress are verified. 2. Experimental 2.1. Reagents and apparatus The CPP-based probe RhB-R12K-FITC was synthesized, high efficiency liquid chromatography (HPLC) purified and characterized with ESI-MS by Shanghai Top-peptide Co., Ltd. (Shanghai, China). The stock solution of the probe was prepared by dissolving the powder in ultrapure water to 1 mM. Glutathione (GSH), N-Ethylmaleimide (NEM), N-
Corresponding author. E-mail address: [email protected] (S. Zhang).
http://dx.doi.org/10.1016/j.talanta.2017.09.044 Received 4 July 2017; Received in revised form 7 September 2017; Accepted 16 September 2017 Available online 22 September 2017 0039-9140/ © 2017 Elsevier B.V. All rights reserved.
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for another 30 min. Fluorescence imaging experiments were performed on a FV1000 confocal laser scanning microscope. The excitation wavelength of LysoTracker Blue DND-22 was 405 nm and the fluorescence signal was collected from 425 nm to 465 nm. The excitation wavelength of RhB-R12K-FITC was 488 nm. The fluorescence signal of FITC part was collected from 505 nm to 540 nm and RhB part was collected from 575 nm to 655 nm. HeLa cells were incubated with RhB-R12K-FITC (2 μM) in DMEM without phenol red at 37 °C for 50 min. The original medium was removed and the cells were washed twice with PBS. Then, HeLa cells were incubated with DMEM containing MitoTracker Deep Red (100 nM) for another 30 min. Fluorescence imaging experiments were performed on a FV1000 confocal laser scanning microscope. The excitation wavelength of RhB-R12K-FITC was 488 nm. The fluorescence signal of FITC part was collected from 505 nm to 540 nm and RhB part was collected from 580 nm to 615 nm. The excitation wavelength of MitoTracker Deep Red FM was 635 nm and the fluorescence signal was collected from 655 nm to 755 nm.
acetylcysteine (NAC), chloroquine and all metal chlorides of analytical grade were purchased from Sigma Aldrich and used without further purification·H2O2 was obtained from Beijing Chemical Works (Beijing, China). Nigericin was purchased from J & K Chemical Technology (Beijing, China). Dulbecco's modified eagle media (DMEM), Dulbecco's modified eagle media without phenol red, fetal bovine serum (FBS), penicillin and streptomycin (100 U/mL), Trypsin EDTA and phosphate buffered saline (PBS) solution were purchased from GIBICO (Invitrogen, USA). Organelle specific dyes LysoTracker Blue DND-22 and MitoTracker Deep Red FM were purchased from Molecular Probes (Invitrogen, USA). 3-(4, 5-dimethyl-2-thiazolyl)−2, 5-diphenyl-2-Htetrazolium bromide (MTT) and Albumin Bovine V (BSA) were purchased from Biodee Biotechnology (Beijing, China). Ultrapure water (over 18 kΩ) from Milli-Q water purification system (Millipore) was used throughout the experiment. HeLa cells were obtained from Peking Union Medical College Hospital (Beijing, China). The absorption spectra were recorded with a U-3900 spectrophotometer (Hitachi, Japan). The Fluorescence spectra of RhB-R12K-FITC at varied pH values and the interference of different species to the ratio of the probe were recorded with a F-7000 fluorescence spectrometer (Hitachi, Japan). The quantum yields were measured by a FLS920 steady state and transient state fluorescence spectrometer (Edinburgh Instruments, UK). The absorbance of MTT assay was measured by using a microplate reader M3 (Molecular Devices). Fluorescence imaging experiments were performed on a FV1000 confocal laser scanning microscope (Olympus, Japan) with a 60× objective lens. HeLa cells were incubated in a MCO5AC CO2 incubator (Panasonic, Japan). All buffer solutions were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd. (Beijing, China). B-R buffer solutions at different pH values were achieved by adding NaOH or HCl to the mixture of 40 mM acetic acid, phosphoric acid and boric acid. High K+ buffer solutions were made up of 120 mM KCl, 30 mM NaCl, 20 mM HEPES, 5 mM glucose, 1 mM CaCl2, 1 mM NaH2PO4 and 0.5 mM MgSO4.
2.5. Intracellular pH calibration HeLa cells were incubated with RhB-R12K-FITC (2 μM) in DMEM without phenol red at 37 °C. After 50 min, the original medium was removed and the cells were washed with PBS three times. Then, the cells were incubated with high K+ buffer solutions in the presence of nigericin (10 μM) at varied pH values in the incubator for 15 min. The fluorescence images were collected and analyzed with Olympus software (FV10-ASW). The fluorescence signal of FITC part was collected from 505 nm to 540 nm and RhB part was collected from 580 nm to 615 nm. The pH calibration curve was constructed according to the ratios of the selected regions of interest (ROIs), which were calculated pixel-by-pixel. All data were expressed as mean ± standard deviation. 2.6. Drug stimulation
2.2. Fluorescence spectral properties of RhB-R12K-FITC HeLa cells were incubated with RhB-R12K-FITC (2 μM) in DMEM without phenol red at 37 °C for 50 min. The medium was removed and the cells were treated with NAC (1 mM) for another 1 h. Fluorescence imaging experiments were performed on FV1000 confocal laser scanning microscope. The fluorescence signal of FITC part was collected from 505 nm to 540 nm and RhB part was collected from 580 nm to 615 nm. The pH values of lysosomes in selected ROIs were determined by the pH calculated curve. All data were expressed as mean ± standard deviation. HeLa cells treated with NEM (1 mM), chloroquine (200 μM) and H2O2 (100 μM) followed the same procedure.
RhB-R12K-FITC (2 μM) in B-R buffer solutions at varied pH values were used for pH calibration curve. Briefly, 2 μL RhB-R12K (1 mM) and 998 μL B-R buffer solutions at specific pH value were mixed and the fluorescence spectra were recorded by a fluorescence spectrometer. The excitation wavelength was 488 nm. The pH value of RhB-R12K-FITC (4 μM) solution between 5 and 8 was adjusted back and forth by 5 M NaOH or HCl solution. The interference of redox species (0.1 mM H2O2, 1 mM GSH, 1 mM NAC and 1 mM NEM), BSA (10 μM), Na+ (1 mM), K+ (1 mM) and some other metal ions (Ca2+, Mg2+, Mn2+, Cu2+, Fe3+, Fe2+, Al3+, Zn2+, Co2+, Cr3+, Ba2+) (100 μM) to the relative ratio of RhB-R12K-FITC (2 μM) at pH 4.9 and pH 7.4 was investigated, respectively. Briefly, 2 μL RhB-R12K (1 mM) was added to 998 μL buffer solutions containing different metal ions. The fluorescence spectra were recorded by using a fluorescence spectrometer. The excitation wavelength was 488 nm.
2.7. MTT assay HeLa cells were seeded in 96-well microtiter plates at a density of 8000 cells/well and cultured at 37 °C in a 5% CO2 incubator for 24 h. The medium was removed and replaced with DMEM added the CPPbased probe RhB-R12K-FITC (2 μM). The cells were incubated with RhBR12K-FITC for 1 h, 3 h, 6 h and 24 h, respectively. Then, 100 μL of the MTT solution (0.5 mg/mL) was added to each well. After 4 h, the MTT solution was abandoned and 100 μL of DMSO was added to each well to dissolve the formed formazan. The plates were shaken for 10 min and the absorbance at 490 nm was measured by a microplate reader M3.
2.3. Cell culture HeLa cells were cultured in DMEM medium supplemented with 10% FBS, 100 U/mL 1% penicillin and streptomycin (v/v) at 37 °C in a 5% CO2 incubator. Cells were seeded in 15 mm confocal laser culture dishes and cultured in the medium for 24 h. The original medium was removed and cells were washed twice with PBS (pH = 7.4) before use.
3. Results and discussion
2.4. Colocalization analysis
3.1. Synthesis and characterization of RhB-R12K-FITC
HeLa cells were incubated with RhB-R12K-FITC (2 μM) in DMEM without phenol red at 37 °C for 50 min. The original medium was removed and the cells were washed twice with PBS. Then, HeLa cells were incubated with DMEM containing LysoTracker Blue DND-22 (50 nM)
The synthetic route of RhB-R12K-FITC was outlined in Scheme 1. Rhodamine B (RhB) and fluorescein isothiocyanate (FITC) was conjugated to R12K in DMF with the catalysis of O-benzotriazole-N, N, N′, N′-tetramethyluronium hexafluorophosphate (HBTU) and N356
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Scheme 1. Synthetic routes of RhB-R12K-FITC and proposed mechanism for fluorescence changes at varied pH values.
ethyldiisopropylamine (DIEA). The final product was characterized by HPLC (Fig. S1) and ESI-MS (Fig. S2) with the purity over 95%. The absorption spectra of RhB-R12K-FITC in buffer solutions at pH 4.0 and 8.0 were obtained (Fig. S3). The probe displayed maximum absorption at 564 nm and 507 nm at pH 4.0 and pH 8.0, respectively. The results are in consensus with spectral properties of RhB spirolactam derivative and FITC under similar conditions [34]. Fluorescence properties of the probe were investigated and single
excitation mode (λex = 488 nm) was used in this work. Fig. 1 indicates fluorescence spectra change of this probe in Britton−Robinson (B-R) buffer solutions at varied pH values. RhB spirolactam part and FITC part show the strongest emission wavelength at 582 nm and 519 nm, respectively. The fluorescence intensity of the RhB spirolactam part decreased with the increase of pH values, which is contrary to that of FITC part. The pKa values of the two parts are provided in the Supporting Information (Fig. S4). The ratio of fluorescence intensities
Fig. 1. a) Fluorescence spectra of RhB-R12K-FITC (2 μM) in buffer solutions at varied pH values. b) Plot of pH versus I519/I582. λex = 488 nm. I519 and I582 are the fluorescence intensity of RhB-R12K-FITC at 519 nm and 582 nm, respectively.
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Fig. 2. a) Reversible fluorescence spectra and b) reversible ratio (F519/F582) changes of RhB-R12K-FITC (4 μM) between pH 4 and 8 in B-R buffer solutions. λex = 488 nm. I519 and I582 are the fluorescence intensity of RhB-R12K-FITC at 519 nm and 582 nm, respectively.
Fig. 3. Colocalization experiment of RhB-R12K-FITC and organelle-specific dyes performed on a confocal laser scanning microscope. (a-e) HeLa cells co-incubated with RhB-R12K-FITC (2 μM) and LysoTracker Blue DND-22 (50 nM). a) LysoTracker Blue DND-22 excited at 405 nm and collected from 425 nm to 465 nm. RhB-R12K-FITC was excited at 488 nm. b) FITC channel and c) RhB channel were collected at 505–540 nm and 575–655 nm, respectively. d) Overlay of (a) and (c). e) Overlay of (a) and (b). (f-j) Hela cells co-incubated with RhB-R12KFITC (2 μM) and MitoTracker Deep Red (100 nM). f) MitoTracker Deep Red excited at 635 nm and collected at 655–755 nm. RhB-R12K-FITC was excited at 488 nm. g) FITC channel and h) RhB channel were collected at 505–540 nm and 580–615 nm, respectively. i) Overlay of (f) and (h). j) Overlay of (f) and (g). Scale bar: 20 µm.
3.2. Spectral characterization of RhB-R12K-FITC in HeLa cells
F519/F582 increased from 0.2 to 9.2 when the pH values increased from 3.3 to 8.1. As proved, the ratio values (F519/F582) have no change with concentration (Fig. S5). The pKa value of RhB-R12K-FITC was calculated to be 5.30 ± 0.02. The quantum yields of the RhB spirolactam and FITC at varied pH values were listed in Table S1. The selectivity, reversibility and cytotoxicity of the probe were studied respectively. We measured and calculated the relative ratio values of the probe in the presence of some common metal ions, redox species and Albumin Bovine V (BSA) under different pH conditions. Fig. S6 and S7 show that these species have little interference to the ratio values, which implies that RhB-R12K-FITC is suitable for intracellular pH measurement. Moreover, the fluorescence spectra and the ratio values (F519/F582) show favourable reversibility between pH 4 and 8 (Fig. 2), which was also an advantageous characteristic for measurement of intracellular pH fluctuation. The cytotoxicity of RhB-R12K-FITC was evaluated by the standard MTT assay. The cell viability determined by the absorbance of MTT at 490 nm confirmed that the probe exhibited low toxicity to Hela cells (Fig. S8).
The arginine-rich CPP R12K endows the probe with excellent membrane permeability and wide dispersion in live cells, which make RhB-R12K-FITC suitable for intracellular pH measurement. Fluorescence imaging of Hela cells was conducted on a confocal microscope. The fluorescence spectra of RhB-R12K-FITC at pH 4.0 and 7.9 in live cells were measured (Fig. S9) and the fluorescence emission maxima were at 581 nm and 511 nm, respectively. Fig. S9c indicates that the FITC part and RhB part had distinguishable fluorescence spectrum and negligible overlap in HeLa cells. 3.3. Colocalization analysis Colocalization analysis was performed. The intracellular locations of the two different fluorescence emission of RhB-R12K-FITC were determined by the organelle-specific dyes LysoTracker Blue DND-22 and MitoTracker Deep Red FM. As shown in Fig. 3, the fluorescence emission of RhB part mainly overlaps with the lysosome specific dye 358
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Fig. 4. Fluorescent images of RhB-R12K-FITC (2 μM) in Hela cells clamped at pH 4.6 (a-d), 5.5 (e-h), 6.1 (i-l), 7.0 (m-p) and 7.9 (q-t). The first row (FITC channel) and the second row (RhB channel) were collected at 505–540 nm and 580–615 nm, respectively. The images on the third row were corresponding contrast images. The ratio images (fourth row) were obtained by calculations of FITC channel and RhB channel. λex = 488 nm. Scale bar: 30 µm.
LysoTracker Blue DND-22. The fluorescence emission of the FITC part spread over the whole cell (including lysosomes) and cannot be localized to the specific organelle. The reasonable explanation for the phenomenon is that the CPP-based probes are delivered into cells by endocytosis and partly release from endosomes after internalization. RhB spirolactam emits strong fluorescence in lysosomes and endosomes for the ring-opening reaction under acidic conditions. FITC emits strong fluorescence in whole cell due to the high concentration of the probe in acidified organelles and the basic pH of cytoplasm and nucleus.
intracellular pH to the surrounding culture medium. Fig. 4 illustrates that the fluorescence intensity of the FITC part increases with pH values, whereas the RhB part has an opposite tendency. The ratio images were provided in the fourth row of Fig. 4. A liner calibration curve (Fig. 5a) from pH 4.6–7.9 (R2 = 0.9944) was obtained. The ratio values increased 98 folds over the pH range. Intracellular pH mapping of the intact HeLa cells was realized (Fig. 5b and S10). The ratio values in lysosomes (where RhB part emits strong fluorescence) are significantly lower than other regions and there is little difference between ratio values of cytoplasm and nucleus. According to the intracellular pH calibration curve, the pH values of intact lysosomes, cytoplasm and nucleus were calculated to be 5.3 ± 0.1, 7.5 ± 0.2 and 7.4 ± 0.1, respectively (Fig. 5a).
3.4. Intracellular pH calibration RhB-R12K-FITC was applied to quantify intracellular pH in HeLa cells. The intracellular calibration experiment was conducted in high K+ buffer solutions with nigericin. HeLa cells were clamped at desired pH values by the H+/K+ ionophore nigericin which homogenized 359
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Fig. 5. The influence of chloroquine and redox species on intracellular pH values. a) Plot of ratio values (R) versus pH values (intracellular pH calibration curve). HeLa cells were incubated with RhB-R12K-FITC (2 μM) for 50 min. The original medium was removed. Then, HeLa cells were respectively incubated with DMEM containing b) none, c) chloroquine (200 μM), d) NAC (1 mM), e) NEM (1 mM) and f) H2O2 (100 μM) for another 1 h. RhB-R12K-FITC was excited at 488 nm. FITC channel and RhB channel were collected at 505–540 nm and 580–615 nm, respectively. Ratio images (b-f) were obtained by calculating the intensity of FITC channel and RhB channel. The changes of lysosomal and cytosolic pH values were determined by the intracellular calibration curve. Scale bar: 30 µm.
Acidic and neutral-basic cellular compartments are distinguished in fluorescent images. Intracellular pH fluctuations associated with reductive environment and oxidative stress were also successfully measured. The oxidents were found to cause cytosolic acidification accompanied with lysosomal alkalization, which improves our undestanding of H+ redistribution between different cellular compartments in physiological process. Furthermore, the fluorophores and the CPP of the probe can be replaced to design a series of fluorescent probe for multiple ion detection and subcellular localization analysis. We believe that the CPP-based probe have great applications in live-cell imaging.
3.5. Drug stimulation Chloroquine known as a lysosomotropic base has been reported to block autophagy and increase lysosomal pH [35,36]. Here, RhB-R12KFITC was employed to detect intracellular pH changes stimulated by chloroquine. From Fig. 5c, we can see a significant increase in the ratio value of lysosomes and a simultaneous slight decrease in that of cytoplasm. The lysosomal and cytosolic pH values of HeLa cells treated with chloroquine was calculated to be 6.6 ± 0.1 and 7.3 ± 0.1, respectively. The result indicates the potential of RhB-R12K-FITC to be applied in monitoring pH fluctuations of different cellular compartments. The influence of redox species NAC (GSH precursor), NEM (GSH inhibitor) and H2O2 to intracellular pH fluctuations was investigated. Lysosomal pH values (red) and Cytosolic pH values (blue) of the treated HeLa cells were calculated and marked in Fig. 5a (From left to right: 5.1 ± 0.1, 5.9 ± 0.2, 6.8 ± 0.2, 7.2 ± 0.3, 7.2 ± 0.2, and 7.5 ± 0.1). NAC and NEM were used to regulate intracellular GSH level. Compared to the intact cells in Fig. 5b, HeLa cells incubated with the reductant NAC have no obvious change in intracellular pH values (Fig. 5d) and the calculations indicate little lysosomal and cytosolic acidification. On the contrary, the oxidants, NEM and H2O2 can tremendously change intracellular pH. Fig. 5e and f illustrate dramatic cytosolic acidification and lysosomal alkalization, which agrees the results in literature [6,37]. Simultaneous pH measurement of lysosomes and cytoplasm contributes to intensively understanding the redistribution of H+ between acidified organelles and cytosolic compartments caused by oxidative stress. In comparison to the previously reported ratiometric pH probes [19,21,38], RhB-R12K-FITC provides more precise spatial information about the intracellular pH change.
Acknowledgements We acknowledge the financial support provided by the National Natural Science Foundation of China (21390413 and 21621003), and the 973 program (2013CB933804). Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta.2017.09.044. References [1] J.R. Casey, S. Grinstein, J. Orlowski, Sensors and regulators of intracellular pH, Nat. Rev. Mol. Cell Biol. 11 (2010) 50–61. [2] J. Stinchcombe, G. Bossi, G.M. Griffiths, Linking albinism and immunity: the secrets of secretory lysosomes, Science 305 (2004) 55–59. [3] S. Ohkuma, B. Poole, Fluorescence probe measurement of intralysosomal ph in living cells and perturbation of ph by various agents, PNAS 75 (1978) 3327–3331. [4] A. Roos, W.F. Boron, Intracellular pH, Physiol. Rev. 61 (1981) 296–434. [5] J. Han, K. Burgess, Fluorescent Indicators for Intracellular pH, Chem. Rev. 110 (2010) 2709–2728. [6] D.S. Kaufman, M.S. Goligorsky, E.P. Nord, M.L. Graber, Perturbation of cell ph regulation by H2O2 in renal epithelial-cells, Arch. Biochem. Biophys. 302 (1993) 245–254. [7] K. Kagedal, M. Zhao, I. Svensson, U.T. Brunk, Sphingosine-induced apoptosis is dependent on lysosomal proteases, Biochem. J. 359 (2001) 335–343. [8] C. Nilsson, U. Johansson, A.C. Johansson, K. Kagedal, K. Ollinger, Cytosolic acidification and lysosomal alkalinization during TNF-alpha induced apoptosis in U937 cells, Apoptosis 11 (2006) 1149–1159.
4. Conclusions A CPP-based ratiometric pH probe RhB-R12K-FITC has been constructed for intracellular pH mapping and simultaneous detection of cytosolic and lysosomal pH change in live cell. This new CPP-based fluorescent probe shares the advantages of small organic fluorophores and CPPs. Benefiting from its wide distribution in live cell, more precise spatial information about intracellular pH distribution can be provided. 360
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A cell-penetrating ratiometric probe for simultaneous measurement of lysosomal and cytosolic pH change
MARK
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Meng-Chan Xia, Lesi Cai, Sichun Zhang , Xinrong Zhang Department of Chemistry, Beijing Key Laboratory of Microanalytical Methods and Instrumentation, Tsinghua University, Beijing 100084, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords: Intracellular pH Fluorescent probes Sensors Cell imaging Cell-penetrating peptides
A new ratiometric fluorescent probe based on cell-penetrating peptides (CPPs) was constructed for whole-cell pH mapping and simultaneous measurement of pH changes in the cytoplasm and lysosomes. The arginine-rich CPP, R12K worked as linker, carrier and part of the fluorophore. Benefiting from R12K, the fluorescent probe is completely water soluble, membrane permeable and well biocompatible. It shows high selectivity, sensitivity and reversibility to pH fluctuations. The ratio of fluorescence intensities F519/F582 increased from 0.2 to 9.2 over the pH range from 3.3 to 8.1. Intracellular pH mapping was successfully realized owing to the wide distribution of the probe in live cells (even in nucleus). Moreover, cytosolic and lysosomal pH change caused by the stimuli can be simultaneously detected. Compared to other ratiometric pH probes, RhB-R12K-FITC can provide more precise information about H+ redistribution between different cellular compartments.
1. Introduction Cells are highly compartmentalized and organized. Different cellular compartments with varied pH values provide distinct conditions for optimal operation [1]. The lumens of lysosomes and endosomes are faintly acidic [2,3], whereas nucleus and cytoplasm are near neutral [4,5]. The pH changes of lysosomes and cytoplasm are closely related in basic cellular activities, such as metabolism [1], oxidative stress [6] and apoptosis [7,8]. Abnormal pH values can lead to cellular dysfunction and indicate the risk of cancer [9–11], neurodegenerative disorders [12,13] and cardiovascular disease [14]. Monitoring intracellular pH homeostasis and how pH is regulated can improve our understanding of physiological and pathological processes. Fluorescent imaging has occupied a decisive position in live cell imaging attributed to their high selectivity, excellent sensitivity and spatiotemporal resolution [15–18]. Ratiometric fluorescent probes give more accurate analysis on intracellular pH, because of the corrections of some systematic errors resulted from dye leakage, photobleaching and optical path length. Although various small-molecule probes and nanoprobes were developed for ratiometric pH sensing [19–27], most of them focus on one specific organelle. For in-depth research on H+ redistribution in physiological process and cellular behavior, simultaneous pH analysis of different cellular compartments is urgently required. Using a combination of multiple organelle-specific pH sensors is much more complicated [28] and researchers are devoted to developing effective pH indicators for intracellular pH mapping [19,21,29]. As far
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as we know, few pH probes allow simultaneous detection of cytosolic and lysosomal pH change. Cell penetrating peptides (CPPs) have been promising carriers to facilitate cellular internalization [30,31]. Our group previously reported a spirolactam derivative RhB-R12K by conjugating the fluorescent dye rhodamine B to CPP [32]. Here, we fully took advantage of the remaining active groups in the CPP and constructed a ratiometric fluorescent pH probe RhB-R12K-FITC. The new probe, which shows high selectivity and sensitivity to H+, possesses excellent membrane permeability, water-solubility and low toxicity. It can even be phagocytized by nucleus due to the typical feature of arginine-rich CPPs [33]. Its remarkable properties and wide distribution in live cells enable relatively successful intracellular pH mapping. The pH value of specific compartment can be measured individually. Furthermore, the pH fluctuations and H+ redistribution between cytoplasm and lysosomes caused by oxidative stress are verified. 2. Experimental 2.1. Reagents and apparatus The CPP-based probe RhB-R12K-FITC was synthesized, high efficiency liquid chromatography (HPLC) purified and characterized with ESI-MS by Shanghai Top-peptide Co., Ltd. (Shanghai, China). The stock solution of the probe was prepared by dissolving the powder in ultrapure water to 1 mM. Glutathione (GSH), N-Ethylmaleimide (NEM), N-
Corresponding author. E-mail address: [email protected] (S. Zhang).
http://dx.doi.org/10.1016/j.talanta.2017.09.044 Received 4 July 2017; Received in revised form 7 September 2017; Accepted 16 September 2017 Available online 22 September 2017 0039-9140/ © 2017 Elsevier B.V. All rights reserved.
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for another 30 min. Fluorescence imaging experiments were performed on a FV1000 confocal laser scanning microscope. The excitation wavelength of LysoTracker Blue DND-22 was 405 nm and the fluorescence signal was collected from 425 nm to 465 nm. The excitation wavelength of RhB-R12K-FITC was 488 nm. The fluorescence signal of FITC part was collected from 505 nm to 540 nm and RhB part was collected from 575 nm to 655 nm. HeLa cells were incubated with RhB-R12K-FITC (2 μM) in DMEM without phenol red at 37 °C for 50 min. The original medium was removed and the cells were washed twice with PBS. Then, HeLa cells were incubated with DMEM containing MitoTracker Deep Red (100 nM) for another 30 min. Fluorescence imaging experiments were performed on a FV1000 confocal laser scanning microscope. The excitation wavelength of RhB-R12K-FITC was 488 nm. The fluorescence signal of FITC part was collected from 505 nm to 540 nm and RhB part was collected from 580 nm to 615 nm. The excitation wavelength of MitoTracker Deep Red FM was 635 nm and the fluorescence signal was collected from 655 nm to 755 nm.
acetylcysteine (NAC), chloroquine and all metal chlorides of analytical grade were purchased from Sigma Aldrich and used without further purification·H2O2 was obtained from Beijing Chemical Works (Beijing, China). Nigericin was purchased from J & K Chemical Technology (Beijing, China). Dulbecco's modified eagle media (DMEM), Dulbecco's modified eagle media without phenol red, fetal bovine serum (FBS), penicillin and streptomycin (100 U/mL), Trypsin EDTA and phosphate buffered saline (PBS) solution were purchased from GIBICO (Invitrogen, USA). Organelle specific dyes LysoTracker Blue DND-22 and MitoTracker Deep Red FM were purchased from Molecular Probes (Invitrogen, USA). 3-(4, 5-dimethyl-2-thiazolyl)−2, 5-diphenyl-2-Htetrazolium bromide (MTT) and Albumin Bovine V (BSA) were purchased from Biodee Biotechnology (Beijing, China). Ultrapure water (over 18 kΩ) from Milli-Q water purification system (Millipore) was used throughout the experiment. HeLa cells were obtained from Peking Union Medical College Hospital (Beijing, China). The absorption spectra were recorded with a U-3900 spectrophotometer (Hitachi, Japan). The Fluorescence spectra of RhB-R12K-FITC at varied pH values and the interference of different species to the ratio of the probe were recorded with a F-7000 fluorescence spectrometer (Hitachi, Japan). The quantum yields were measured by a FLS920 steady state and transient state fluorescence spectrometer (Edinburgh Instruments, UK). The absorbance of MTT assay was measured by using a microplate reader M3 (Molecular Devices). Fluorescence imaging experiments were performed on a FV1000 confocal laser scanning microscope (Olympus, Japan) with a 60× objective lens. HeLa cells were incubated in a MCO5AC CO2 incubator (Panasonic, Japan). All buffer solutions were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd. (Beijing, China). B-R buffer solutions at different pH values were achieved by adding NaOH or HCl to the mixture of 40 mM acetic acid, phosphoric acid and boric acid. High K+ buffer solutions were made up of 120 mM KCl, 30 mM NaCl, 20 mM HEPES, 5 mM glucose, 1 mM CaCl2, 1 mM NaH2PO4 and 0.5 mM MgSO4.
2.5. Intracellular pH calibration HeLa cells were incubated with RhB-R12K-FITC (2 μM) in DMEM without phenol red at 37 °C. After 50 min, the original medium was removed and the cells were washed with PBS three times. Then, the cells were incubated with high K+ buffer solutions in the presence of nigericin (10 μM) at varied pH values in the incubator for 15 min. The fluorescence images were collected and analyzed with Olympus software (FV10-ASW). The fluorescence signal of FITC part was collected from 505 nm to 540 nm and RhB part was collected from 580 nm to 615 nm. The pH calibration curve was constructed according to the ratios of the selected regions of interest (ROIs), which were calculated pixel-by-pixel. All data were expressed as mean ± standard deviation. 2.6. Drug stimulation
2.2. Fluorescence spectral properties of RhB-R12K-FITC HeLa cells were incubated with RhB-R12K-FITC (2 μM) in DMEM without phenol red at 37 °C for 50 min. The medium was removed and the cells were treated with NAC (1 mM) for another 1 h. Fluorescence imaging experiments were performed on FV1000 confocal laser scanning microscope. The fluorescence signal of FITC part was collected from 505 nm to 540 nm and RhB part was collected from 580 nm to 615 nm. The pH values of lysosomes in selected ROIs were determined by the pH calculated curve. All data were expressed as mean ± standard deviation. HeLa cells treated with NEM (1 mM), chloroquine (200 μM) and H2O2 (100 μM) followed the same procedure.
RhB-R12K-FITC (2 μM) in B-R buffer solutions at varied pH values were used for pH calibration curve. Briefly, 2 μL RhB-R12K (1 mM) and 998 μL B-R buffer solutions at specific pH value were mixed and the fluorescence spectra were recorded by a fluorescence spectrometer. The excitation wavelength was 488 nm. The pH value of RhB-R12K-FITC (4 μM) solution between 5 and 8 was adjusted back and forth by 5 M NaOH or HCl solution. The interference of redox species (0.1 mM H2O2, 1 mM GSH, 1 mM NAC and 1 mM NEM), BSA (10 μM), Na+ (1 mM), K+ (1 mM) and some other metal ions (Ca2+, Mg2+, Mn2+, Cu2+, Fe3+, Fe2+, Al3+, Zn2+, Co2+, Cr3+, Ba2+) (100 μM) to the relative ratio of RhB-R12K-FITC (2 μM) at pH 4.9 and pH 7.4 was investigated, respectively. Briefly, 2 μL RhB-R12K (1 mM) was added to 998 μL buffer solutions containing different metal ions. The fluorescence spectra were recorded by using a fluorescence spectrometer. The excitation wavelength was 488 nm.
2.7. MTT assay HeLa cells were seeded in 96-well microtiter plates at a density of 8000 cells/well and cultured at 37 °C in a 5% CO2 incubator for 24 h. The medium was removed and replaced with DMEM added the CPPbased probe RhB-R12K-FITC (2 μM). The cells were incubated with RhBR12K-FITC for 1 h, 3 h, 6 h and 24 h, respectively. Then, 100 μL of the MTT solution (0.5 mg/mL) was added to each well. After 4 h, the MTT solution was abandoned and 100 μL of DMSO was added to each well to dissolve the formed formazan. The plates were shaken for 10 min and the absorbance at 490 nm was measured by a microplate reader M3.
2.3. Cell culture HeLa cells were cultured in DMEM medium supplemented with 10% FBS, 100 U/mL 1% penicillin and streptomycin (v/v) at 37 °C in a 5% CO2 incubator. Cells were seeded in 15 mm confocal laser culture dishes and cultured in the medium for 24 h. The original medium was removed and cells were washed twice with PBS (pH = 7.4) before use.
3. Results and discussion
2.4. Colocalization analysis
3.1. Synthesis and characterization of RhB-R12K-FITC
HeLa cells were incubated with RhB-R12K-FITC (2 μM) in DMEM without phenol red at 37 °C for 50 min. The original medium was removed and the cells were washed twice with PBS. Then, HeLa cells were incubated with DMEM containing LysoTracker Blue DND-22 (50 nM)
The synthetic route of RhB-R12K-FITC was outlined in Scheme 1. Rhodamine B (RhB) and fluorescein isothiocyanate (FITC) was conjugated to R12K in DMF with the catalysis of O-benzotriazole-N, N, N′, N′-tetramethyluronium hexafluorophosphate (HBTU) and N356
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Scheme 1. Synthetic routes of RhB-R12K-FITC and proposed mechanism for fluorescence changes at varied pH values.
ethyldiisopropylamine (DIEA). The final product was characterized by HPLC (Fig. S1) and ESI-MS (Fig. S2) with the purity over 95%. The absorption spectra of RhB-R12K-FITC in buffer solutions at pH 4.0 and 8.0 were obtained (Fig. S3). The probe displayed maximum absorption at 564 nm and 507 nm at pH 4.0 and pH 8.0, respectively. The results are in consensus with spectral properties of RhB spirolactam derivative and FITC under similar conditions [34]. Fluorescence properties of the probe were investigated and single
excitation mode (λex = 488 nm) was used in this work. Fig. 1 indicates fluorescence spectra change of this probe in Britton−Robinson (B-R) buffer solutions at varied pH values. RhB spirolactam part and FITC part show the strongest emission wavelength at 582 nm and 519 nm, respectively. The fluorescence intensity of the RhB spirolactam part decreased with the increase of pH values, which is contrary to that of FITC part. The pKa values of the two parts are provided in the Supporting Information (Fig. S4). The ratio of fluorescence intensities
Fig. 1. a) Fluorescence spectra of RhB-R12K-FITC (2 μM) in buffer solutions at varied pH values. b) Plot of pH versus I519/I582. λex = 488 nm. I519 and I582 are the fluorescence intensity of RhB-R12K-FITC at 519 nm and 582 nm, respectively.
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Fig. 2. a) Reversible fluorescence spectra and b) reversible ratio (F519/F582) changes of RhB-R12K-FITC (4 μM) between pH 4 and 8 in B-R buffer solutions. λex = 488 nm. I519 and I582 are the fluorescence intensity of RhB-R12K-FITC at 519 nm and 582 nm, respectively.
Fig. 3. Colocalization experiment of RhB-R12K-FITC and organelle-specific dyes performed on a confocal laser scanning microscope. (a-e) HeLa cells co-incubated with RhB-R12K-FITC (2 μM) and LysoTracker Blue DND-22 (50 nM). a) LysoTracker Blue DND-22 excited at 405 nm and collected from 425 nm to 465 nm. RhB-R12K-FITC was excited at 488 nm. b) FITC channel and c) RhB channel were collected at 505–540 nm and 575–655 nm, respectively. d) Overlay of (a) and (c). e) Overlay of (a) and (b). (f-j) Hela cells co-incubated with RhB-R12KFITC (2 μM) and MitoTracker Deep Red (100 nM). f) MitoTracker Deep Red excited at 635 nm and collected at 655–755 nm. RhB-R12K-FITC was excited at 488 nm. g) FITC channel and h) RhB channel were collected at 505–540 nm and 580–615 nm, respectively. i) Overlay of (f) and (h). j) Overlay of (f) and (g). Scale bar: 20 µm.
3.2. Spectral characterization of RhB-R12K-FITC in HeLa cells
F519/F582 increased from 0.2 to 9.2 when the pH values increased from 3.3 to 8.1. As proved, the ratio values (F519/F582) have no change with concentration (Fig. S5). The pKa value of RhB-R12K-FITC was calculated to be 5.30 ± 0.02. The quantum yields of the RhB spirolactam and FITC at varied pH values were listed in Table S1. The selectivity, reversibility and cytotoxicity of the probe were studied respectively. We measured and calculated the relative ratio values of the probe in the presence of some common metal ions, redox species and Albumin Bovine V (BSA) under different pH conditions. Fig. S6 and S7 show that these species have little interference to the ratio values, which implies that RhB-R12K-FITC is suitable for intracellular pH measurement. Moreover, the fluorescence spectra and the ratio values (F519/F582) show favourable reversibility between pH 4 and 8 (Fig. 2), which was also an advantageous characteristic for measurement of intracellular pH fluctuation. The cytotoxicity of RhB-R12K-FITC was evaluated by the standard MTT assay. The cell viability determined by the absorbance of MTT at 490 nm confirmed that the probe exhibited low toxicity to Hela cells (Fig. S8).
The arginine-rich CPP R12K endows the probe with excellent membrane permeability and wide dispersion in live cells, which make RhB-R12K-FITC suitable for intracellular pH measurement. Fluorescence imaging of Hela cells was conducted on a confocal microscope. The fluorescence spectra of RhB-R12K-FITC at pH 4.0 and 7.9 in live cells were measured (Fig. S9) and the fluorescence emission maxima were at 581 nm and 511 nm, respectively. Fig. S9c indicates that the FITC part and RhB part had distinguishable fluorescence spectrum and negligible overlap in HeLa cells. 3.3. Colocalization analysis Colocalization analysis was performed. The intracellular locations of the two different fluorescence emission of RhB-R12K-FITC were determined by the organelle-specific dyes LysoTracker Blue DND-22 and MitoTracker Deep Red FM. As shown in Fig. 3, the fluorescence emission of RhB part mainly overlaps with the lysosome specific dye 358
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Fig. 4. Fluorescent images of RhB-R12K-FITC (2 μM) in Hela cells clamped at pH 4.6 (a-d), 5.5 (e-h), 6.1 (i-l), 7.0 (m-p) and 7.9 (q-t). The first row (FITC channel) and the second row (RhB channel) were collected at 505–540 nm and 580–615 nm, respectively. The images on the third row were corresponding contrast images. The ratio images (fourth row) were obtained by calculations of FITC channel and RhB channel. λex = 488 nm. Scale bar: 30 µm.
LysoTracker Blue DND-22. The fluorescence emission of the FITC part spread over the whole cell (including lysosomes) and cannot be localized to the specific organelle. The reasonable explanation for the phenomenon is that the CPP-based probes are delivered into cells by endocytosis and partly release from endosomes after internalization. RhB spirolactam emits strong fluorescence in lysosomes and endosomes for the ring-opening reaction under acidic conditions. FITC emits strong fluorescence in whole cell due to the high concentration of the probe in acidified organelles and the basic pH of cytoplasm and nucleus.
intracellular pH to the surrounding culture medium. Fig. 4 illustrates that the fluorescence intensity of the FITC part increases with pH values, whereas the RhB part has an opposite tendency. The ratio images were provided in the fourth row of Fig. 4. A liner calibration curve (Fig. 5a) from pH 4.6–7.9 (R2 = 0.9944) was obtained. The ratio values increased 98 folds over the pH range. Intracellular pH mapping of the intact HeLa cells was realized (Fig. 5b and S10). The ratio values in lysosomes (where RhB part emits strong fluorescence) are significantly lower than other regions and there is little difference between ratio values of cytoplasm and nucleus. According to the intracellular pH calibration curve, the pH values of intact lysosomes, cytoplasm and nucleus were calculated to be 5.3 ± 0.1, 7.5 ± 0.2 and 7.4 ± 0.1, respectively (Fig. 5a).
3.4. Intracellular pH calibration RhB-R12K-FITC was applied to quantify intracellular pH in HeLa cells. The intracellular calibration experiment was conducted in high K+ buffer solutions with nigericin. HeLa cells were clamped at desired pH values by the H+/K+ ionophore nigericin which homogenized 359
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Fig. 5. The influence of chloroquine and redox species on intracellular pH values. a) Plot of ratio values (R) versus pH values (intracellular pH calibration curve). HeLa cells were incubated with RhB-R12K-FITC (2 μM) for 50 min. The original medium was removed. Then, HeLa cells were respectively incubated with DMEM containing b) none, c) chloroquine (200 μM), d) NAC (1 mM), e) NEM (1 mM) and f) H2O2 (100 μM) for another 1 h. RhB-R12K-FITC was excited at 488 nm. FITC channel and RhB channel were collected at 505–540 nm and 580–615 nm, respectively. Ratio images (b-f) were obtained by calculating the intensity of FITC channel and RhB channel. The changes of lysosomal and cytosolic pH values were determined by the intracellular calibration curve. Scale bar: 30 µm.
Acidic and neutral-basic cellular compartments are distinguished in fluorescent images. Intracellular pH fluctuations associated with reductive environment and oxidative stress were also successfully measured. The oxidents were found to cause cytosolic acidification accompanied with lysosomal alkalization, which improves our undestanding of H+ redistribution between different cellular compartments in physiological process. Furthermore, the fluorophores and the CPP of the probe can be replaced to design a series of fluorescent probe for multiple ion detection and subcellular localization analysis. We believe that the CPP-based probe have great applications in live-cell imaging.
3.5. Drug stimulation Chloroquine known as a lysosomotropic base has been reported to block autophagy and increase lysosomal pH [35,36]. Here, RhB-R12KFITC was employed to detect intracellular pH changes stimulated by chloroquine. From Fig. 5c, we can see a significant increase in the ratio value of lysosomes and a simultaneous slight decrease in that of cytoplasm. The lysosomal and cytosolic pH values of HeLa cells treated with chloroquine was calculated to be 6.6 ± 0.1 and 7.3 ± 0.1, respectively. The result indicates the potential of RhB-R12K-FITC to be applied in monitoring pH fluctuations of different cellular compartments. The influence of redox species NAC (GSH precursor), NEM (GSH inhibitor) and H2O2 to intracellular pH fluctuations was investigated. Lysosomal pH values (red) and Cytosolic pH values (blue) of the treated HeLa cells were calculated and marked in Fig. 5a (From left to right: 5.1 ± 0.1, 5.9 ± 0.2, 6.8 ± 0.2, 7.2 ± 0.3, 7.2 ± 0.2, and 7.5 ± 0.1). NAC and NEM were used to regulate intracellular GSH level. Compared to the intact cells in Fig. 5b, HeLa cells incubated with the reductant NAC have no obvious change in intracellular pH values (Fig. 5d) and the calculations indicate little lysosomal and cytosolic acidification. On the contrary, the oxidants, NEM and H2O2 can tremendously change intracellular pH. Fig. 5e and f illustrate dramatic cytosolic acidification and lysosomal alkalization, which agrees the results in literature [6,37]. Simultaneous pH measurement of lysosomes and cytoplasm contributes to intensively understanding the redistribution of H+ between acidified organelles and cytosolic compartments caused by oxidative stress. In comparison to the previously reported ratiometric pH probes [19,21,38], RhB-R12K-FITC provides more precise spatial information about the intracellular pH change.
Acknowledgements We acknowledge the financial support provided by the National Natural Science Foundation of China (21390413 and 21621003), and the 973 program (2013CB933804). Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta.2017.09.044. References [1] J.R. Casey, S. Grinstein, J. Orlowski, Sensors and regulators of intracellular pH, Nat. Rev. Mol. Cell Biol. 11 (2010) 50–61. [2] J. Stinchcombe, G. Bossi, G.M. Griffiths, Linking albinism and immunity: the secrets of secretory lysosomes, Science 305 (2004) 55–59. [3] S. Ohkuma, B. Poole, Fluorescence probe measurement of intralysosomal ph in living cells and perturbation of ph by various agents, PNAS 75 (1978) 3327–3331. [4] A. Roos, W.F. Boron, Intracellular pH, Physiol. Rev. 61 (1981) 296–434. [5] J. Han, K. Burgess, Fluorescent Indicators for Intracellular pH, Chem. Rev. 110 (2010) 2709–2728. [6] D.S. Kaufman, M.S. Goligorsky, E.P. Nord, M.L. Graber, Perturbation of cell ph regulation by H2O2 in renal epithelial-cells, Arch. Biochem. Biophys. 302 (1993) 245–254. [7] K. Kagedal, M. Zhao, I. Svensson, U.T. Brunk, Sphingosine-induced apoptosis is dependent on lysosomal proteases, Biochem. J. 359 (2001) 335–343. [8] C. Nilsson, U. Johansson, A.C. Johansson, K. Kagedal, K. Ollinger, Cytosolic acidification and lysosomal alkalinization during TNF-alpha induced apoptosis in U937 cells, Apoptosis 11 (2006) 1149–1159.
4. Conclusions A CPP-based ratiometric pH probe RhB-R12K-FITC has been constructed for intracellular pH mapping and simultaneous detection of cytosolic and lysosomal pH change in live cell. This new CPP-based fluorescent probe shares the advantages of small organic fluorophores and CPPs. Benefiting from its wide distribution in live cell, more precise spatial information about intracellular pH distribution can be provided. 360
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