Differential vital staining of normal fibroblasts
and melanoma cells by an anionic conjugated
polyelectrolyte
Karin Magnusson, Hanna Appelqvist, Artur Cieslar-Pobuda, Jens Wigenius, Thommie Karlsson, Marek Jan Los, Bertil Kågedal, Jon Jonasson and K.P Nilsson
Linköping University Post Print
N.B.: When citing this work, cite the original article.
Original Publication:
Karin Magnusson, Hanna Appelqvist, Artur Cieslar-Pobuda, Jens Wigenius, Thommie Karlsson, Marek Jan Los, Bertil Kågedal, Jon Jonasson and K.P Nilsson, Differential vital staining of normal fibroblasts and melanoma cells by an anionic conjugated polyelectrolyte, 2015, Cytometry Part A, (87), 3, 262-272.
http://dx.doi.org/10.1002/cyto.a.22627 Copyright: Wiley
http://eu.wiley.com/WileyCDA/
Postprint available at: Linköping University Electronic Press http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-115887
1 #14-120-R1
Differential vital staining of normal fibroblasts and melanoma cells by an
anionic conjugated polyelectrolyte
Short title: Differential PTAA staining in normal fibroblasts and melanoma cells
Karin Magnusson 1, Hanna Appelqvist 1, Artur Cieślar-Pobuda 2, 3, Jens Wigenius 4£, Thommie Karlsson 5§, Marek J. Łos 2, 6*, Bertil Kågedal 7, Jon Jonasson 8 and K. Peter R. Nilsson 1*
1
Division of Chemistry, Department of Physics, Chemistry and Biology, Linköping University, Linköping, Sweden
2 Division of Cell Biology, and Integrative Regenerative Medicine Center (IGEN), Department Clinical and Experimental Medicine (IKE), Linköping University, Sweden
3 Institute of Automatic Control, Silesian University of Technology, Gliwice, Poland 4
Biomolecular and Organic Electronics, Department of Physics, Chemistry and Biology, Linköping University, Linköping, Sweden
5
Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Faculty of Health Science, Linköping University, Linköping, Sweden
6
Department of Pathology, Pomeranian Medical University, Szczecin, Poland 7
Division of Clinical Chemistry, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden
8
Division of Clinical Pathology and Clinical Genetics, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden
£ Current position: Sales Account Manager at Carl Zeiss AB §
Current position: Application Specialist Confocal Microscopy at Leica Microsystems
Keywords: conjugated polyelectrolyte, luminescent conjugated polythiophene, fibroblast,
melanoma, fluorescence, photo-induced toxicity
Abbreviations: CPE, Conjugated polyelectrolyte; LCO, Luminescent conjugated
oligothiophene; LCP, Luminescent conjugated polythiophene; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PTAA, polythiophene acetic acid
2
*Correspondence:
Peter Nilsson
Division of Chemistry
Department of Physics, Chemistry and Biology Linköping University S-581 83 Linköping, Sweden E-mail: petni@ifm.liu.se Phone nr: +46 13 282787 Marek Łos MD/PhD
Department Clinical and Experimental Medicine (IKE)
Integrative Regenerative Medicine Center (IGEN)
Linköping University
Cell Biology Building, Level 10 S-581 85 Linköping, Sweden E-mail: marek.los@liu.se Phone nr: +46 10 10 32787
3
Abstract
Molecular probes for imaging of live cells are of great interest for studying biological and pathological processes. The anionic luminescent conjugated polythiophene (LCP) polythiophene acetic acid (PTAA), has previously been used for vital staining of cultured fibroblasts as well as transformed cells with results indicating differential staining due to cell phenotype. Herein, we investigated the behavior of PTAA in two normal and five transformed cells lines. PTAA fluorescence in normal cells appeared in a peripheral punctuated pattern whereas the probe was more concentrated in a one-sided perinuclear localization in the five transformed cell lines. In fibroblast, PTAA fluorescence was initially associated with fibronectin and after 24 h partially localized to lysosomes. The uptake and intracellular target in malignant melanoma cells was more ambiguous and the intracellular target of PTAA in melanoma cells is still elusive. PTAA was well tolerated by both fibroblasts and melanoma cells, and microscopic analysis as well as viability assays showed no signs of negative influence on growth. Stained cells maintained their proliferation rate for at least 12 generations. Although the probe itself was non-toxic, photo-induced cellular toxicity was observed in both cell lines upon irradiation directly after staining. However, no cytotoxicity was detected when the cells were irradiated 24 h after staining, indicating that the photo-induced toxicity is dependent on the cellular location of the probe. Overall, these studies certified PTAA as a useful agent for vital staining of cells, and that PTAA can potentially be utilized to study cancer-related biological and pathological processes.
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Introduction
Fluorescent molecular probes for identification of various cellular compartments and components in cultured cells offer the possibility to visualize the dynamics and biochemical pathways of pathological and biological processes. From a biophysical perspective, such probes should preferably be highly selective, environmentally sensitive and display high optical stability. A class of molecules that fulfill these characteristics is conjugated polyelectrolytes (CPEs). These probes possess unique electronic and optical properties and have been widely used in optoelectronic devices such as organic light emitting diodes and solar cells (1-6), as well as for fluorescent biosensing of DNA-hybridization (7-10) and protein-ligand interactions (11-14). Lately, CPEs have also been employed as fluorescent reporters in more complex biological environments, such as cells (15-23). Linear CPEs have been used for staining of fibroblasts (15,16) and apoptotic cells (17), whereas grafted CPEs have been reported as a novel class of self-assembled highly fluorescent nanoparticles that can be utilized for cell imaging (18-21). These grafted CPEs can be randomly functionalized by proper ligands to create specificity towards a distinct biological target, such as cancer cells (20). The concept of utilizing CPE based self-assembled highly fluorescent nanoparticles for imaging has also been modified further by the development of hyperbranched CPEs and such molecules have been utilized for specific staining of cancer cells and cytoplasmic proteins in living cells (22,23).
CPEs consisting of a repetitive flexible thiophene backbone possess unique spectral properties, since the conjugated backbone can be twisted or extended and emit light with different colors depending on the conformational restriction of the backbone. Thus, upon interaction with specific targets, distinct optical signatures are achieved from the CPEs, and in
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contrast to antibodies, these molecular probes are preferably recognizing certain structural motifs instead of particular biomolecules. CPEs have been employed for studying disease associated protein aggregates, and luminescent conjugated polythiophenes, LCPs, have evolved as a novel class of ligands for studying pathological hallmarks of protein aggregation diseases such as Alzheimer´s disease and the infectious prion diseases (24-26). Thiophene fluorophores have also been utilized for live staining of cells (27,28) and an anionic LCP, polythiophene acetic acid (PTAA; Fig. 1A) was shown to target acidic vesicles in normal cells (15). However, the targeting could not be demonstrated in transformed cells, which indicates that PTAA staining could be utilized to distinguish normal and malignant cells.
Herein, we investigate the differential staining of normal and transformed cells by PTAA in more detail, as well as the dye´s performance for long term staining of living cells. The present work confirmed that PTAA can be used for specific staining of structures in living cells, and that the staining pattern is dissimilar in human fibroblasts compared to malignant melanoma cells. In addition, PTAA displayed rapid internalization and good retention inside the cells, and did not alter cell viability nor proliferation. The intracellular staining of PTAA in the fibroblasts was, as shown previously (15), to some extent associated to lysosomes, whereas no co-staining was observed with a variety of other cellular compartments. Furthermore, kinetic studies also revealed fibronectin as a potential mediator for internalization of PTAA into the fibroblasts. Overall, these studies verified that PTAA could be utilized for staining of living cells, and highlighted some of its potential applications.
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Materials and Methods
Cells and Culture Conditions
Human lung fibroblasts MRC-5 (CCL-171; ATCC, Manassas, VA, USA) and malignant melanoma cells SK-MEL-28 (HTB-72; ATCC) were cultured in BioAMF-2 complete cell culture medium (Kibbutz Beit Haemek, Israel). Melanocytes were kindly provided by Petra Wäster and cultured as described previously (29). Human cervical cancer cells HeLa (CCL-2; ATCC), breast cancer cells MDA-MB-231 (HTB-26; ATCC) and human colon cancer HCT-116 (CCL-247; ATCC) were cultured in Dulbecco’s modified eagle medium GlutaMAX containing 50 IU/ml penicillin-G, 50 µg/ml streptomycin and 10% fetal bovine serum, all from Gibco (Paisly, UK). Neuroblastoma cells SH-SY5Y (94030304; Sigma-Aldrich, St. Louis, MO, USA) were cultured in Eagle’s minimum essential medium GlutaMAX containing the additions mentioned above. Cells were incubated in humidified air with 5% CO2 at 37°C. The day before experiments, cells were trypsinized and seeded to reach 50% confluence.
Vital staining of cells with PTAA
The synthesis of PTAA has been reported elsewhere (30) and the probe was dissolved in deionized water at a concentration of 1 mg/ml. Cells seeded on coverslips were stained with PTAA (50 µg/ml) in complete medium for 30 min, 37°C. The superfluous probe was removed and the cells were rinsed three times with PBS and incubated in fresh medium for indicated periods of time. For microscopic evaluation, the cells were rinsed three times with PBS, fixed in 4% paraformaldehyde (PFA; 20 min, 4°C), mounted using Vectashield with DAPI (Vector Laboratories, Burlingame, CA, USA) and examined with a Zeiss confocal microscope, LSM
7 780 (Carl Zeiss AG, Oberkochen, Germany).
Flow cytometric analysis
For flow cytometry, the cells were detached by trypzination, washed with PBS and analyzed using a Gallios flow cytometer (Beckman coulter, Gallios™, USA). The fluorescence was measured by using a 488 nm laser and 620/30 BP filter (FL3). 20 000 cells were collected for each sample and the data was analyzed using the software Kaluza.
Staining with PTAA after fixation of cells
Cells seeded on coverslips were rinsed three times with PBS and fixed in methanol, (20 min, -20°C) or in 4% PFA (20 min, 4°C). After three rinses with PBS, the cells were stained with PTAA (50 µg/ml) in PBS for 30 min at room temperature. The cells were then rinsed three times in PBS and mounted using Vectashield (Vector Laboratories) and examined with a Zeiss confocal microscope, LSM 780.
Co-staining with organelle markers
For staining of mitochondria, PTAA-stained cells were incubated with MitoTracker Orange CMTMRos (150 nM, 30 min, 37°C; Molecular Probes, Eugene, OR, USA). Cells were then fixed in 4% PFA (20 min, 4°C). For immunostaining, PTAA-stained cells were after fixation permeabilized with 0.1% saponin (Sigma-Aldrich) in PBS containing 5% fetal bovine serum (20 min, room temperature) and incubated for 2 h at room temperature with one of the following monoclonal mouse primary antibodies: Golga2/GM130 (1:500, Novus Biologicals,
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Littleton, CO, USA), lysosome-associated membrane protein 2 (LAMP-2, 1:50; Southern Biotech, Birmingham, AL, USA), p62 (1:100, BD Biosciences, Franklin Lakes, NJ, USA), or polyclonal anti-rabbit primary antibodies; α-tubulin (1:1000, Abcam, Cambridge, UK), calnexin (1:700, Novus Biologicals), early endosomal antigen-1 (EEA-1, 1:400; Sigma-Aldrich), fibronectin (1:400, Sigma-Sigma-Aldrich), LC3B (1:200, Novus Biologicals), Niemann-Pick type C1 (NPC1, 1:500; Abcam), peroxisomal membrane protein 70 (PMP70, 1:1000; Molecular Probes) and proteasome 20S (1:100, Abcam). This step was followed by incubation with the appropriate secondary antibodies conjugated to Alexa Fluor 594 (1:400, Molecular Probes) for 1 h. All incubations were done in the dark. Next, the cells were mounted in Vectashield (Vector Laboratories) and examined with a Zeiss confocal microscope, LSM 780 (Carl Zeiss AG, Germany).
Fluorescence and spectral analysis of stained cells
Fluorescence and spectral images of stained cells were collected with an inverted Zeiss (Axio Observer.Z1) LSM 780 microscope equipped with a 32 channel QUASAR GaAsP spectral array detector. The differential interference contrast microscopy (DIC) image was collected with an upright Zeiss LSM 700 confocal microscope. A plan-Apochromat 63x/1.40 Oil DIC M27 objective lens was used for all imaging. The different excitation wavelengths for imaging the cells are listed in the figure legends. Emission spectra were collected by simultaneous excitation of two laser lines, one at 405 and one at 561 nm. Each of the four spectra in figure 3K represents normalized and average data from nine areas in three different images for each of the two comparisons; PTAA vs Alexa 594-fibronectin, and PTAA vs Alexa 594-LAMP-2.
9 Viability analysis
The reducing capacity of cell cultures was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay (Sigma-Aldrich). This method is widely used to assess cytotoxicity and cell viability, and it is currently thought that the amount of MTT formazan is proportional to the number of living cells (31). Cells were incubated with 0.5 mg/ml MTT for 2 h at 37°C. Then, the MTT solution was removed and the formazan product was dissolved in dimethyl sulfoxide. The absorbance was measured at 550 nm with a VICTOR X Series Multiple Plate Reader (PerkinElmer, Waltham, MA USA).
Live-cell microscopy
Live-cell studies were done on newly stained cells and on cells 24 h after using a JuLI™ fluorescent cell analyzer (NanoEnTek Inc., Newton, MA, USA) equipped with a White/Blue LED light source. Both the fluorescent light source optimized for green fluorescent protein (GFP) fluorescence (excitation 488 nm, emission 520 nm) and bright field were used. The cells were exposed to light once every minute for 6 h with an illumination intensity of 100% for the fluorescent mode and 10% for the bright field mode. Control experiments were done on stained cells without fluorescent excitation and unstained cells using both fluorescent light source and bright field.
Live/Dead assay
LIVE/DEAD® Viability/Cytotoxicity Kit (Molecular Probes) was used on cells analyzed in JuLI™ for 6 h under the conditions described above. The cells were washed twice in PBS, incubated in solution containing 2 µM Calcein AM and 4 µM Ethidium homodimer-1 in PBS
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in 37°C for 20 min. After washing in PBS, the cells were examined with a Zeiss fluorescence microscope, Axio Vert.A1 (Carl Zeiss AG).
Statistical Analysis
Experiments were routinely performed at least three times, and the results are presented as the means and standard deviations of independent samples. Differences between two groups were analyzed by the two-tailed Student's t-test and, for more than two groups, by one-way ANOVA with the Bonferroni post hoc test. Statistical calculations were performed using the GraphPad Prism 6 software package. Differences were considered significant when p 0.05.
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Results
PTAA staining in normal cells compared to transformed cells
In order to investigate differences between the behavior of PTAA in normal and transformed cells, two normal cell lines (fibroblasts and melanocytes) and five transformed cell lines (melanoma, cervical cancer, breast cancer, colon cancer and neuroblastoma), were stained with PTAA and analyzed in parallel. The cells were stained for 30 min with PTAA and incubated for 24 h in complete medium prior to examination. In agreement with the previous study (15), the pattern of PTAA staining in normal cells was different from that of malignant cells (Fig. 1). After 24 h peripheral punctate staining were observed in fibroblasts and melanocytes (Fig. 1B, C), whereas in the malignant cells the dots were concentrated in a one-sided perinuclear localization (Fig. 1D-H). Since we observed a striking difference in the size of the foci between fibroblasts and melanoma cells, these cell lines were selected for further evaluation of the PTAA staining.
To analyze these essentially different staining patterns further, we investigated the kinetics of the PTAA staining. The cells were stained for 30 min with PTAA, returned to the cell culture medium and incubated for different periods of time (Fig. 2). Directly after staining, PTAA displayed a characteristic filamentous staining pattern associated to the edges, or the plasma membrane of the fibroblasts (Fig. 2A). After further incubation (1 to 6 h), the filamentous staining pattern gradually disappeared, and an intracellular punctate pattern was predominant. The filamentous staining had almost disappeared after 12 h and after 24 h only larger cytosolic foci were visible. When analyzing the PTAA staining pattern in the melanoma cells over time, a pronounced difference was detected compared to the fibroblasts (Fig. 2B). Immediately after staining, PTAA fluorescence was observed in the cytosol and the probe was distributed throughout the entire cell. After 1 h, the fluorescence was redirected to
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small dots found close to one side of the nucleus and, evidently, this pattern did not change over time. The staining pattern after 24 h of incubation was similar to the pattern observed after 1h of incubation.
Flow cytometry analysis was also implemented on PTAA stained cells to obtain more quantitative information about the fluorescence intensity and the kinetics (Fig. 2C-E). The histograms were broader for the fibroblasts, which indicate a larger distribution in fluorescence whereas the melanoma cells were more uniform in their fluorescence. The highest fluorescence intensity according to arithmetic mean (mean value of fluorescence intensity for whole population) was obtained 24 h after staining in both cell lines, even though the intensity 1 h after staining also showed to be significantly different from unstained cells. Overall, the results from the kinetic experiments indicated that both internalization and compartmentalization of PTAA were different in fibroblasts when compared to melanoma cells.
Co-staining experiments
To assess the cellular targets for PTAA at distinct points in time, co-staining experiments with PTAA in combination with fluorescent probes specifically targeting different cellular components were performed. The fluorescent probes were chosen to have distinct emission spectra compared to PTAA and thereby co-localization of PTAA and specific cellular targets could be examined with fluorescence microscopy. As shown in figure 3A, the filamentous extracellular staining pattern, which appeared immediately after PTAA staining of fibroblasts, displayed partial co-localization with fibronectin staining. Some areas of the filamentous network showed both the presence of PTAA (green) and fibronectin (red; Fig. 3A). To verify the probable co-localization, emission spectra were collected from images of cells exposed to
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a combination of two excitation wavelengths, 405 and 561 nm. When comparing the emission spectra from these areas, a mixture of the emission signature from PTAA and the Alexa fluorophore conjugated secondary antibody, directed towards the fibronectin antibody, was achieved (Fig. 3K). In contrast, PTAA displayed no co-localization with α-tubulin, which indicates that this protein is not associated to the fibrillar structure stained by PTAA (Fig. 3B). Differential interference contrast images also verified that PTAA restricted to distinct regions of the plasma membrane (Supplementary Material Fig. S1).
The intracellular cytosolic punctate pattern that was observed in fibroblasts after 24 h of incubation showed no sign of co-localization with Golgi, mitochondria, p62, ER, proteasomes, autophagosomes or peroxisomes (Fig. 3C-I). However, partial co-localization was observed when combining PTAA staining with antibody staining directed to lysosome-associated membrane protein 2 (LAMP-2; Fig. 3J), suggesting that PTAA to some extent is associated to lysosomes. Spectral analysis of these entities also revealed a mixture of the emission signature from PTAA and the Alexa fluorophore used to detect the presence of LAMP-2 (Fig. 3K). Thus, PTAA was partly co-localized with lysosomes, but might also stain a yet unidentified subcellular structure.
Similar co-staining experiments as described above were also performed for PTAA stained malignant melanoma cells (Fig. S2). However, these experiments were not successful as the PTAA staining in the transformed cells was misrepresented after using the permeabilization buffer necessary for achieving consistent antibody staining (Fig. S2 and Fig. 2B). The permeabilization buffers tested contained saponin (0.1%), Triton X (0.2%), Tween (0.05%), NP40 (0.5%), digitonin (500 µg/ml), acetone or methanol. Overall, the PTAA staining pattern that consisted of small foci distributed close to the nucleus was not evident after treating the cells with permeabilization buffers, suggesting that in contrast to what was
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observed in fibroblasts, the PTAA staining in melanoma cells was sensitive to detergents. The punctated pattern in the fibroblasts were maintained after treatment with all above mentioned permeabilization buffers, thus verifying that PTAA stained normal fibroblasts and melanoma cells in different manners.
Uptake of PTAA
To further explore the cellular uptake of PTAA, the PTAA intensity was investigated when cells were stained at 4°C instead of 37°C to see whether the probe enters even at low cell activity. There was still intracellular PTAA staining at low temperature incubation, even though the fluorescence intensity was lower (Fig. 4A). Thus, PTAA is most likely entering cells by a relatively temperature-independent pathway. PTAA was also tested for co-staining with markers for the major components of the endocytic pathway, but showed no co-localization with early or late endosomes (Fig. 4B). These results indicated that endocytosis is not the major uptake mechanism for PTAA in human fibroblasts. When staining was performed after the cells were fixed, the probe stained the entire cells (Fig. S3), as previously reported (15). Thus, the PTAA staining patterns were strikingly different compared to results obtained when staining living cells, which indicates that the filamentous staining pattern and the foci are due to specific cellular processes in living cells.
PTAA staining – influence on cell viability and potential toxicity
As PTAA might be utilized as a potential fluorescent tool for live imaging of cells, the influence of PTAA on cell viability was investigated. Fibroblasts and melanoma cells were stained with PTAA for 30 min, washed and incubated in complete medium for 24 hours and then compared to unstained cells. When studying phase contrast images of the cells, no sign
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of negative influence from PTAA was detected, since the unstained and the stained cells had similar morphology (Fig. 5A). In addition, the PTAA stained cells maintained their proliferation rate, since they divided at the same rate as unstained cells for at least 12 generations. The lack of toxicity was also confirmed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay, where the conversion of soluble MTT into formazan is used as a measure of cell viability (32). The reduction assay verified that formazan production was equal in unstained and stained cells for both cell lines 24 h after staining (Fig. 5B) and thus PTAA staining does not influence cell viability. Overall, PTAA was well tolerated by both cell lines and these results certified that PTAA could potentially be utilized for long-term live imaging of cells.
To explore any potential photo-induced cellular toxicity of PTAA, cells were stained with PTAA, washed and incubated in complete cell medium in a fluorescent cell analyzer JuLI™. Subsequently, the cells were exposed to light using a GFP-optimized excitation. As shown in figure 6A and movie S1, the exposure to light had a major impact on both PTAA stained fibroblasts and melanoma cells. By excitation once every minute for 6 h, the cells shrank and showed apoptotic morphology with rounding of cells, (Fig. 6A, first and second column) whereas the morphology of cells outside the light-exposed area did not change. Unfortunately, the light source was relatively weak and the emission wavelength of the light source was not fitting the emission spectra of the probe, therefore only bright field images are shown. The photo-induced toxicity was also verified with a Live/Dead assay, where live cells can be seen in green and dead cells in red. In the area that had been exposed to light, cells were round and several red cells could be observed (Fig. 6A, third column), whereas outside this area, the proportion of green cells was dramatically increased. Hence, PTAA induced cell death in both normal and transformed cells upon irradiation.
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As PTAA displayed time dependent staining pattern, we next examined if the photo-induced toxicity was dependent on the cellular location of the dye. Analogous to the experiments described above, cells were stained with PTAA for 30 min, washed and incubated in complete cell medium for 24 h, allowing PTAA to accumulate in the cytosolic compartments observed after 24 h (Fig. 1B, D). In contrast to PTAA stained cells that were exposed to irradiation immediately after staining, cells that were stained and then incubated for 24 h prior to light exposure did not change their morphology by light exposure (Fig. 6B, first and second column). It was not possible to locate the area of excitation after exposure since all the cells were unaffected and this observation was also verified with the Live/Dead assay where only green and prosperous looking cells were observed. Hence, photo-induced toxicity was only observed when exposing newly stained cells, suggesting that the cellular location of the probe was determining the photo-induced toxicity.
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Discussion
CPEs have emerged as an interesting class of molecular probes for fluorescent imaging of cells, since these compounds are highly selective, display high optical stability and exhibit unique interaction induced optical properties (15-23). Here, we have shown that an anionic thiophene based CPE, denoted PTAA, can be employed for differential staining of normal and transformed cells, as well as for live imaging of cells. The staining procedure is straightforward and less complex compared to staining techniques using antibodies. In addition, the staining is persistent and does not show signs of quenching. Thus, our results strongly support the notion that CPEs can be utilized as molecular probes for assessment of various components in cultured cells and that these probes could potentially be employed to visualize the dynamics of pathological and biological processes on a cellular level, as well as for differentiating distinct types of cells.
Although, the staining procedure was identical for normal and transformed cells, the difference in PTAA staining between these cell types was striking. Firstly, the staining pattern was differently distributed in normal cells when compared to malignant cells. In fibroblasts, PTAA displayed a filamentous peripheral pattern and an intracellular punctate pattern, whereas perinuclear dots were observed for melanoma cells. Secondly, the final target in the melanoma cells was reached already 1 h after staining, whereas it took more than 12 h for PTAA to accumulate in the intracellular punctate pattern in the fibroblasts. Thirdly, the PTAA staining in the melanoma cells was almost abolished after incubation in different permeabilization buffers, whereas the foci in the fibroblasts were maintained after equivalent treatments. Altogether, these dissimilarities indicate a difference in localization, structure, accessibility or quantity of the PTAA stained cellular target in normal cells as compared to malignant cells. Hence, identifying the cellular targets that are stained by PTAA is of great
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importance and herein we assessed a variety of conventional markers for cellular components in combination with PTAA staining.
In this study, we identified fibronectin as a potential molecular target of PTAA in fibroblasts. Immediately after staining, PTAA was to some extent associated to fibronectin. This staining pattern was only observed in fibroblasts, as the PTAA pattern and the fibronectin pattern did not match in the melanoma cells. A previous study (16) has also shown a similar co-localization with fibronectin and a carboxylated poly(p-phenyleneethynylene) based CPE. In contrast, when utilizing a cationic poly(p-phenyleneethynylene) based CPE for staining of fibroblasts, the interaction with fibronectin was abolished, indicating that the interaction with fibronectin is only achieved with probes having an anionic polyvalent nature (16). Thus, PTAA might interact with fibronectin in a similar fashion as other polyanionic molecules and fibronectin domains display an extended area of positive charges, a location that is involved in binding of polyanionic heparin (33). Fibronectin, a main part of many extracellular matrices, is involved in cell adhesion, cell-to-cell communication as well as differentiation (34,35). The protein is also important for normal cell growth, cell adhesion and often interacts with integrin receptors. The integrins serve as adhesion receptors during tumor cell proliferation, migration and survival (36-38). The probable absence of co-localization between PTAA and fibronectin in the malignant melanoma cells might be due to reduced levels or localization (Fig. 2B and Fig. S2A) of fibronectin in the oncogenically transformed cells (39-42). This reduction has also been correlated with tumorigenicity and malignancy. Thus, as fibronectin participates in an array of essential cancer-related biological and pathological processes, including cancer cell survival, carcinoma development (43) and metastasis (44), the PTAA staining of fibronectin could be employed for further studies of cancer cells and their interactions with tissue components.
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As shown in the kinetic experiments, PTAA was internalized in both fibroblasts and malignant melanoma cells. However, the internalization in melanoma cells did not seem to be associated to fibronectin, as the internalization in fibroblasts seems to be, suggesting that the mechanism of uptake of PTAA is different between the cell types. Polycationic molecules, such as cell penetrating peptides HIV-Tat or polyarginine, are taken up through endocytosis (45), whereas negatively charged molecules are typically not readily transported into living cells and the delay in uptake in fibroblast has also been observed for other anionic CPEs (16). The results reported herein also suggest that endocytosis is not the major uptake mechanisms for PTAA. Firstly, endocytosis requires cell activity and PTAA internalization occurred also at 4°C in both cell lines. Secondly, no co-localization of PTAA staining with endosomes was detected in fibroblasts. A recent study also showed that a cationic CPE entered renal cell carcinoma cells by a temperature-independent non-endocytic pathway (17). As PTAA co-localize with fibronectin, the internalization of PTAA into fibroblast might be mediated through a fibronectin mediated pathway (16), whereas the rapid uptake of PTAA into melanonoma cells might occur in a diffusion manner or via an as yet unidentified molecular transporter (17). However, further studies on a variety of cell lines are required to elucidate the molecular mechanism underlying the difference in uptake of PTAA in normal and transformed cells.
After internalization, the staining pattern of PTAA, as well as the location, was also different between fibroblasts and melanoma cells. In addition, the filamentous staining pattern and the foci are observed due to specific cellular processes in living cells, since the PTAA staining pattern was completely altered when staining PFA fixed cells. A previous study, utilizing CPEs on mildly fixed cells, indicated that lysosome-related organelles were targeted by both anionic, cationic and zwitterionic polythiophene derivatives in cultured primary cells,
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whereas the targeting of these vesicles could not be demonstrated in transformed cells (15). In the present study, some of the internalized PTAA in the fibroblast co-localized with the lysosomal marker LAMP-2. The intracellular cytosolic punctate pattern in the fibroblasts showed no sign of co-localization with Golgi, mitochondria, p62, ER, proteasomes, autophagosomes or peroxisomes, although as seen in figure 3 some sort of mutually exclusive localization relation was observed between the Golgi complex (Fig. 3C), ER (Fig. 3F) and the PTAA dots, which seemed to be capping the Golgi apparatus. Thus, even though PTAA might stain a yet unidentified subcellular structure, we conclude that PTAA in fibroblasts is to some extent associated with lysosome-related organelles. In contrast, the intracellular target of PTAA in melanoma cells is still elusive since the PTAA staining pattern with small foci distributed close to the nucleus was sensitive to detergents. It is tempting to speculate that these foci may represent PTAA bound to less protected subcellular structures trapped within the Golgi-centrosome complex when comparing the localization of Golgi in figure 3C and the localization of PTAA in figures 1D and 2B. However, the detergent sensitivity precluded a formal proof of this conjecture using antibodies against Golgi.
A potential fluorescent tool for live imaging of cells should preferably be well tolerated by cells and PTAA displayed no negative effect on cell viability in both cell lines. Microscopic analysis as well as viability assay did not show any signs of cellular toxicity induced by PTAA, since the cells continued to divide for more than 12 generations without losing their proliferation rate compared to unstained cells. The lack of cytotoxicity was shown both for normal fibroblasts as well as melanoma cells, and other studies (27,28) have also revealed that oligothiophene derivatives are relatively well tolerated by cells, implicating that thiophene based probes can be utilized as efficient molecular agents for live imaging of cells. However, even though the probe itself is not toxic to the cells, toxicity might be induced by
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external factors such as exposure to light. Photo-induced toxicity can for instance indicate a connection to photodynamic therapy (PDT) where a non-toxic light-sensitive compound localized to a tumor is exposed to light at a specific wavelength and generates cytotoxic species to kill the tumor cells (46). Herein, we observed that cells exposed to light (488 nm) immediately after PTAA staining (0h) shrunk and started to die. In contrast, cells exposed in a similar fashion 24 h after staining did not alter their morphology, indicating that the cellular location of the probe is essential for the photo-induced toxicity. The photo-induced toxicity of PTAA could be observed as a consequence of conformational rearrangement of the probe upon exposure to light. CPEs can undergo optically or electronically induced conformational rearrangement and electronic release of cells grown on a CPE coated surface was recently presented (47). Alternatively, PTAA could potentially act as a photosensitizer that induces cytotoxic species upon exposure to light. It has been demonstrated that lysosome disruption is not directly cytotoxic (48,49) and that PDT treatment of lysosomes does not necessarily affect cell viability (49). However, other studies (50-52) have also suggested that hydrolases and acids leaking out of damaged lysosomes may degrade cellular components and induce cell death. The prospect of utilizing thiophene probes for PDT towards cancer cells is tantalizing, but the mechanism underlying the photo-induced toxicity needs to be investigated further. In addition, a thiophene based probe that selectively stains tumor-associated cells would be desirable. Lately, chemically defined luminescent conjugated oligothiophenes, LCOs, have been developed (53-55) and LCOs with diverse imidazole motifs along the thiophene backbone were identified as an interesting class of agents for staining of cancer cells (55). Thus, it will be of great interest to study their impact and target more in detail within different cell types, as well as their potential for being utilized for PDT.
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can be used for vital staining of cultured cells. The staining pattern was different in the two normal cell types compared to the five transformed cell lines studied here, which suggests a difference in localization, structure, accessibility or quantity of the PTAA stained cellular target in normal cells compared to transformed cells. We foresee that PTAA and other similar thiophene derivatives will be used as additional molecular tools for studying cell-associated biological and pathological processes, as well as for fluorescent imaging of living cells.
Acknowledgment
Our work is supported by the Swedish Foundation for Strategic Research (K.P.R.N., K.M., HA). K.P.R.N is financed by an ERC Starting Independent Researcher Grant (Project: MUMID) from the European Research Council. M.J.L. kindly acknowledge the core/startup support from Linkoping University, from Integrative Regenerative Medicine Center (IGEN), from Cancerfonden (2013/391), and from VR-NanoVision (K2012-99X -22325-01-5). A.C.P. acknowledges support form BK/265/RAU1/2014/t.10. K.M is enrolled in the doctoral program Forum Scientum. Grateful acknowledges is directed to Petra Wäster for providing melanocyte cultures.
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References
1. Kim JY, Lee K, Coates NE, Moses D, Nguyen TQ, Dante M, Heeger AJ. Efficient Tandem Polymer Solar Cells Fabricated by All-Solution Processing. Science 2007;317:222–225. 2. Hoven CV, Garcia A, Bazan GC, Nguyen TQ. Recent Applications of Conjugated Polyelectrolytes in Optoelectronic Devices. Adv Mater Weinheim 2008;20:3793–3810. 3. Huang F, Wu H, Cao Y. Water/alcohol soluble conjugated polymers as highly efficient electron transporting/injection layer in optoelectronic devices. Chem Soc Rev 2010;39:2500– 2521.
4. Taranekar P, Qiao Q, Jiang H, Ghiviriga I, Schanze KS, Reynolds JR. Hyperbranched Conjugated Polyelectrolyte Bilayers for Solar-Cell Applications. J Am Chem Soc 2007;129:8958–8959.
5. Liu X, Zhu R, Zhang Y, Bin Liu, Ramakrishna S. Anionic benzothiadiazole containing polyfluorene and oligofluorene as organic sensitizers for dye -sensitized solar cells. Chemical Communications 2008;0:3789–3791.
6. Zhang W, Fang Z, Su M, Saeys M, Liu B. A Triphenylamine‐ Based Conjugated Polymer with Donor‐ π‐ Acceptor Architecture as Organic Sensitizer for Dye‐ Sensitized Solar Cells. Macromolecular Rapid Communications 2009;30:1533–1537.
7. Ho HA, Boissinot M, Bergeron MG, Corbeil G, Doré K, Boudreau D, Leclerc M. Colorimetric and fluorometric detection of nucleic acids using cationic polythiophene derivatives. Angew Chem Int Ed Engl 2002;41:1548–1551.
8. Chen L, McBranch DW, Wang H-L, Helgeson R, Wudl F, Whitten DG. Highly sensitive biological and chemical sensors based on reversible fluorescence quenching in a conjugated polymer. PNAS 1999;96:12287–12292.
9. Gaylord BS, Heeger AJ, Bazan GC. DNA detection using water-soluble conjugated polymers and peptide nucleic acid probes. PNAS 2002;99:10954–10957.
10. Nilsson KPR, s OIA. Chip and solution detection of DNA hybridization using a luminescent zwitterionic polythiophene derivative. Nature Materials 2003;2:419–424. 11. Wang D, Gong X, Heeger PS, Rininsland F, Bazan GC, Heeger AJ. Biosensors from conjugated polyelectrolyte complexes. PNAS 2002;99:49–53.
12. Ho HA, Leclerc M. Optical Sensors Based on Hybrid Aptamer/Conjugated Polymer Complexes. J Am Chem Soc 2004;126:1384–1387.
13. Nilsson KPR, Rydberg J, Baltzer L, Inganäs O. Self-assembly of synthetic peptides control conformation and optical properties of a zwitterionic polythiophene derivative. PNAS 2003;100:10170–10174.
24
14. Nilsson KPR, Inganäs O. Optical Emission of a Conjugated Polyelectrolyte: Calcium-Induced Conformational Changes in Calmodulin and Calmodulin−Calcineurin Interactions. Macromolecules 2004;37:9109–9113.
15. Björk P, Nilsson KPR, Lenner L, Kågedal B, Persson B, Inganäs O, Jonasson J.
Conjugated polythiophene probes target lysosome-related acidic vacuoles in cultured primary cells. Molecular and Cellular Probes 2007;21:329–337.
16. McRae RL, Phillips RL, Kim I-B, Bunz UHF, Fahrni CJ. Molecular Recognition Based on Low-Affinity Polyvalent Interactions: Selective Binding of a Carboxylated Polymer to Fibronectin Fibrils of Live Fibroblast Cells. J Am Chem Soc 2008;130:7851–7853. 17. Liu L, Yu M, Duan X, Wang S. Conjugated polymers as multifunctional biomedical platforms: Anticancer activity and apoptosis imaging. J Mater Chem 2010;20:6942. 18. Pu K-Y, Liu B. Fluorescent Conjugated Polyelectrolytes for Bioimaging. Adv Funct Mater 2011;21:3408–3423.
19. Feng X, Tang Y, Duan X, Liu L, Wang S. Lipid -modified conjugated polymer nanoparticles for cell imaging and transfection. J Mater Chem 2010;20:1312–1316. 20. Pu K-Y, Li K, Liu B. A Molecular Brush Approach to Enhance Quantum Yield and Suppress Nonspecific Interactions of Conjugated Polyelectrolyte for Targeted
Far‐ Red/Near‐ Infrared Fluorescence Cell Imaging. Adv Funct Mater 2010;20:2770–2777. 21. Pu K-Y, Cai L, Bin Liu. Design and Synthesis of Charge-Transfer-Based Conjugated Polyelectrolytes as Multicolor Light-Up Probes. Macromolecules 2009;42:5933–5940. 22. Pu K-Y, Li K, Shi J, Bin Liu. Fluorescent Single-Molecular Core−Shell Nanospheres of Hyperbranched Conjugated Polyelectrolyte for Live-Cell Imaging. Chemistry of Materials 2009;21:3816–3822.
23. Li K, Pu K-Y, Cai L, Bin Liu. Phalloidin-Functionalized Hyperbranched Conjugated Polyelectrolyte for Filamentous Actin Imaging in Living Hela Cells. Chemistry of Materials 2011;23:2113–2119.
24. Nilsson KPR, Herland A, Hammarström P, Inganäs O. Conjugated polyelectrolytes: conformation-sensitive optical probes for detection of amyloid fibril formation. Biochemistry 2005;44:3718–3724.
25. Nilsson KPR, Hammarström P, Ahlgren F, Herland A, Schnell EA, Lindgren M,
Westermark GT, Inganäs O. Conjugated Polyelectrolytes—Conformation‐ Sensitive Optical Probes for Staining and Characterization of Amyloid Deposits. ChemBioChem 2006;7:1096– 1104.
26. Sigurdson CJ, Nilsson KPR, Hornemann S, Manco G, Polymenidou M, Schwarz P, Leclerc M, m PHO, thrich KWU, Aguzzi A. Prion strain discrimination using luminescent conjugated polymers. Nature Methods 2007;4:1023–1030.
25
27. Palamà I, Di Maria F, Viola I, Fabiano E, Gigli G, Bettini C, Barbarella G. Live-cell-permeant thiophene fluorophores and cell-mediated formation of fluorescent fibrils. J Am Chem Soc 2011;133:17777–17785.
28. Di Maria F, Palamà IE, Baroncini M, Barbieri A, Bongini A, Bizzarri R, Gigli G, Barbarella G. Live cell cytoplasm staining and selective labeling of intracellular proteins by non-toxic cell-permeant thiophene fluorophores. Org Biomol Chem 2014;12:1603–1610. 29. Andersson E, Vahlquist A, Rosdahl I. β-Carotene uptake and bioconversion to retinol differ between human melanocytes and keratinocytes. Nutrition and cancer 2001.
30. Ding L, Jonforsen M, Roman LS, Andersson MR. Photovoltaic cells with a conjugated polyelectrolyte. Synthetic metals 2000;110:133–140.
31. van Meerloo J, Kaspers GJL, Cloos J. Cell sensitivity assays: the MTT assay. Methods Mol. Biol. 2011;731:237–245.
32. Denizot F, Lang R. Rapid colorimetric assay for cell growth and survival. Journal of Immunological Methods 1986;89:271–277.
33. Sharma A, Askari JA, Humphries MJ, Jones EY, Stuart DI. Crystal structure of a heparin- and integrin-binding segment of human fibronectin. EMBO J. 1999;18:1468–1479.
34. Mao Y, Schwarzbauer JE. Fibronectin fibrillogenesis, a cell-mediated matrix assembly process. Matrix Biology 2005;24:389–399.
35. Boudreau NJ, Jones PL. Extracellular matrix and integrin signalling: the shape of things to come. Biochem. J. 1999;339 ( Pt 3):481–488.
36. Desgrosellier JS, Cheresh DA. Integrins in cancer: biological implications and therapeutic opportunities. Nat. Rev. Cancer 2010;10:9–22.
37. Mitra SK, Schlaepfer DD. Integrin-regulated FAK-Src signaling in normal and cancer cells. Curr. Opin. Cell Biol. 2006;18:516–523.
38. Guo W, Giancotti FG. Integrin signalling during tumour progression. Nat Rev Mol Cell Biol 2004;5:816–826.
39. Hynes RO. Cell surface proteins and malignant transformation. Biochim Biophys Acta 1976;458:73–107.
40. Vaheri A, Mosher DF. High molecular weight, cell surface-associated glycoprotein (fibronectin) lost in malignant transformation. Biochim Biophys Acta 1978;516:1–25. 41. Yamada KM, Olden K. Fibronectins - adhesive glycoproteins of cell surface and blood. Nature 1978;275:179–184.
42. Wagner DD, Ivatt R, Destree AT, Hynes RO. Similarities and differences between the fibronectins of normal and transformed hamster cells. J Biol Chem 1981;256:11708–11715.
26
43. Han S, Khuri FR, Roman J. Fibronectin stimulates non-small cell lung carcinoma cell growth through activation of Akt/mammalian target of rapamycin/S6 kinase and inactivation of LKB1/AMP-activated protein kinase signal pathways. Cancer Res 2006;66:315–323. 44. Reticker-Flynn NE, Malta DFB, Winslow MM, Lamar JM, Xu MJ, Underhill GH, Hynes RO, Jacks TE, Bhatia SN. A combinatorial extracellular matrix platform identifies cell-extracellular matrix interactions that correlate with metastasis. Nat Commun 2012;3:1122. 45. Joliot A, Prochiantz A. Transduction peptides: from technology to physiology. Nat Cell Biol 2004;6:189–196.
46. Jensen TJ, Vicente MGH, Luguya R, Norton J, Fronczek FR, Smith KM. Effect of overall charge and charge distribution on cellular uptake, distribution and phototoxicity of cationic porphyrins in HEp2 cells. J Photochem Photobiol B, Biol 2010;100:100–111.
47. Persson KM, Karlsson R, Svennersten K, Löffler S, Jager EWH, Richter-Dahlfors A, Konradsson P, Berggren M. Electronic control of cell detachment using a self-doped conducting polymer. Adv Mater Weinheim 2011;23:4403–4408.
48. Okada CY, Rechsteiner M. Introduction of macromolecules into cultured mammalian cells by osmotic lysis of pinocytic vesicles. Cell 1982;29:33–41.
49. Berg K, Moan J. Lysosomes as photochemical targets. Int J Cancer 1994;59:814–822. 50. Geze M, Morliere P, Maziere JC, Smith KM, Santus R. Lysosomes, a key target of hydrophobic photosensitizers proposed for photochemotherapeutic applications. J Photochem Photobiol B, Biol 1993;20:23–35.
51. De Duve C, De Barsy T, Poole B, Trouet A, Tulkens P, Van Hoof FO. Lysosomotropic agents. Biochemical Pharmacology 1974;23:2495–2531.
52. Aits S, Jäättelä M. Lysosomal cell death at a glance. J Cell Sci 2013;126:1905–1912. 53. Aslund A, Sigurdson CJ, Klingstedt T, Grathwohl S, Bolmont T, Dickstein DL, Glimsdal E, Prokop S, Lindgren M, Konradsson P, Holtzman DM, Hof PR, Heppner FL, Gandy S, Jucker M, Aguzzi A, Hammarström P, Nilsson KPR. Novel pentameric thiophene derivatives for in vitro and in vivo optical imaging of a plethora of protein aggregates in cerebral
amyloidoses. ACS Chem Biol 2009;4:673–684.
54. Klingstedt T, Aslund A, Simon RA, Johansson LBG, Mason JJ, Nyström S,
Hammarström P, Nilsson KPR. Synthesis of a library of oligothiophenes and their utilization as fluorescent ligands for spectral assignment of protein aggregates. Org Biomol Chem 2011;9:8356–8370.
55. Cieślar-Pobuda A, Bäck M, Magnusson K, Jain MV, Rafat M, Ghavami S, Nilsson KPR, Los MJ. Cell type related differences in staining with pentameric thiophene derivatives. Cytometry A 2014;85:628-635.
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Figure legends
Figure 1. Fluorescence images of PTAA staining of normal cells compared to transformed
cells. (A) The chemical structure of PTAA (poly thiophene acetic acid), wt = 3 kDa. (B) Human fibroblasts (MRC-5), (C) melanocytes, (D) melanoma cells (SK-MEL-28), (E) human cervical cancer cells (HeLa), (F) breast cancer cells (MDA-MB-231), (G) human colon cancer (HCT-116) and (H) neuroblastoma cells (SH-SY5Y) were stained with PTAA (50 µg/ml; 30 min) and incubated for 24 h in complete medium before fixation and microscopic examination. PTAA staining (green) and cell nuclei labeled with DAPI (blue) were analyzed using lasers at 405 nm and 505 nm. Scale bars 20 µm.
Figure 2. Kinetic studies of PTAA staining. Fluorescence images (A) human fibroblasts
(MRC-5) and (B) melanoma cells (SK-MEL-28) stained with PTAA (50 µg/ml; 30 min; green) and incubated for indicated times in complete medium before microscopic examination. Cell nuclei were labeled with DAPI (blue) and arrowheads mark filamentous staining and arrows mark dots. The cells were analyzed with lasers at 405 nm and 505 nm. Scale bars 20 µm. Flow cytometry analysis of fluorescence intensity in (C) MRC-5 and (D) SK-MEL-28 stained with PTAA as described above. E) Schematic plot of the arithmetic mean and SD of the fluorescence intensity calculated from C) and D) (n=4). Significant differences were determined by one-way ANOVA with the Bonferroni post hoc test and are indicated with asterisks in the figure. * p ≤0.05, ** p≤0.01, *** p≤0.001, **** p≤0.0001
Figure 3. Fluorescence images of PTAA staining in combination with various cellular
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green) and incubated for 0 h (A, B) or 24 h (C – J) in complete medium. Co-localization was analyzed by fluorescent staining of major cellular compartments. (A) fibronectin, (B) -tubulin, (C) Golgi (Golga2), (D) mitochondria (Mitotracker), (E) p62, (F) ER (Calnexin), (G) proteasome 20S, (H) autophagosomes (LC3), (I) peroxisomes (PMP70), (J) lysosomes (LAMP-2), (K) emission spectra of PTAA and Alexa fluorophores visualizing fibronectin and lysosomes. Green arrowheads and arrows show PTAA-staining, red arrowheads and arrows show fluorescent probes targeting different cell compartments. Orange arrowhead and yellow arrow show overlap between PTAA and fibronectin and lysosomes respectively. Co-localization with mitochondria was analyzed with lasers at 488 nm and 550 nm, others with lasers at 488 nm and 595 nm. Scale bars 20 µm.
Figure 4. Fluorescence images of PTAA uptake. (A) Human fibroblasts (MRC-5) and
melanoma cells (SK-MEL-28) were stained with PTAA (50 µg/ml; 30 min) (green) and incubated for 30 min at 37C or at 4C. DAPI (blue) labels cell nucleus. (B) Human fibroblasts (MRC-5) were stained with PTAA (50 µg/ml; 30 min) (green) and incubated for indicated times in complete medium. Co-localization was analyzed by fluorescent staining (red) of early endosomes (EEA1) and late endosomes (NPC1). In A lasers at 405 nm and 488 nm were used, in B 488 nm and 595 nm. Scale bars 20 µm.
Figure 5. Cell morphology and viability after PTAA staining. (A) Human fibroblasts
(MRC-5) and melanoma cells (SK-MEL-28) were stained with PTAA (50 µg/ml; 30 min) washed and incubated in complete medium. Phase contrast images were taken of unstained and stained cells 24 h after staining. Scale bars 50 µm. (B) The reducing capacity of cell cultures
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at 24 h as assessed by the MTT assay (n=4). The reducing capacity can be denoted as a measure of cell viability. Data are presented as mean + SD.
Figure 6. Images of PTAA-mediated photo-induced cellular toxicity. Human fibroblasts
(MRC-5) and melanoma cells (SK-MEL-28) were stained with PTAA (50 µg/ml; 30 min) washed and incubated in complete medium. Light exposure using a GFP-optimized fluorescence light source (excitation 488 nm, emission 520 nm) once every minute for 6 h was performed (A) immediately after staining and (B) 24 h after staining. First column shows phase contrast images of cells before light exposure. Second column shows phase contrast images of cells after light exposure. Third column shows fluorescence image of cells after light exposure and after assessment with the Live/Dead assay (live cells in green and dead cells in red). The images were recorded using lasers at 495 nm and 585 nm. Scale bars 100 µm.
