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Further insights into the assessment of cell cycle phases by FTIR microspectroscopy
Vibrational Spectroscopy 75 (2014) 127–135
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Vibrational Spectroscopy journal homepage: www.elsevier.com/locate/vibspec
Further insights into the assessment of cell cycle phases by FTIR microspectroscopy Diana E. Bedolla a , Saˇsa Kenig a , Elisa Mitri b , Paola Storici a , Lisa Vaccari a,∗ a b
Elettra – Sincrotrone Trieste, S.S. 14 Km 163.5, 34149 Basovizza, Trieste, Italy CNR-IOM, TASC Laboratory, S.S. 14 Km 163.5, 34149 Basovizza, Trieste, Italy
a r t i c l e
i n f o
Article history: Available online 1 September 2014 Keywords: FTIR microspectroscopy Flow cytometry Cell cycle B16 mouse melanoma cells U2OS human bone osteosarcoma cells
a b s t r a c t Cell growth and replication occurs in an orderly manner through a set of tightly coordinated physiological events, classified as G0, G1, S, G2 and M in conformity to their characteristics. In a previous work, by combining the results of flow cytometry (FC) using propidium iodide (PI) staining, PI-FC, and Fourier Transform Infrared Microspectroscopy (FTIRM), we gathered information to classify live B16 cells into three different set of phases (G0/G1, S and G2/M), according to their nucleic acid content measured as the area integral of the Phosphate I band (PhI, 1274–1182 cm−1 ). In this work, we demonstrate that, once built a calibration dataset for a cell line determining the intervals of the PhI area integral related to each phase of the cell cycle, such data can be used for assigning the stage to which a live cell belongs without the support of FC. In addition, we evaluate the spectral profile of early G1 B16 cells, and compare it with the one of G0 and late G1 cell cycle phases. FTIRM highlights that G0 and G1 phases are a continuum, where the content of RNA of early G1 cells is in between G0 and late G1, and the overall nucleic acid content varies accordingly. In the paper, we also pinpoint the effects on synchronization protocols on cellular biochemistry, further strengthening the potentialities of a totally label-free methodology for cell sorting. Finally, we demonstrate that the general concept behind the proposed approach may be extended to other mammalian cell lines: human bone osteosarcoma (U2OS) cells were tested. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Eukaryotic cells follow an ordered set of growth and division events known as cell cycle [1]. The cell cycle is divided into four main steps, called Gap 1 (G1), Synthesis (S), Gap 2 (G2) and Mitosis (M), each of them characterized by peculiar cellular activities [2]. The G1 phase, also called growth phase, starts right after the mitotic process and is characterized by protein and mRNA synthesis as well as by production of more cell organelles; the volume of the cytoplasm increases and the cell size follows the same trend. G1 stage is further divided into early G1 and late G1, sometimes also called G1A and G1B [3]. Cells in late G1 have higher content of both RNA and proteins compared to post-mitotic early G1 cells, and can directly enter the S phase, while cells in early G1 need to increase their RNA concentration above a threshold level to be able to initiate DNA replication (restriction point). In the following S phase,
Abbreviations: FTIRM, Fourier Transform Infrared Microspectroscopy; FC, flow cytometry; PI, propidium iodine; NA, nucleic acid. ∗ Corresponding author. Tel.: +39 040 3758567; fax: +39 040 9380902. E-mail address: [email protected] (L. Vaccari). http://dx.doi.org/10.1016/j.vibspec.2014.08.007 0924-2031/© 2014 Elsevier B.V. All rights reserved.
the cell commits to DNA replication. At the end of the S phase, the amount of DNA in the cell is doubled and two identical set of chromosomes are produced. The G2 phase lasts until the cell enters in mitosis and it is characterized by unaffected DNA content while an intense bio-synthetic activity takes place. In five consecutive stages of M phase (prophase, prometaphase, metaphase, anaphase, telophase/cytokinesis) nuclear membrane disintegrates, DNA condenses and chromosomes are separated and split into two equal portions. In the end, the cell divides into two identical daughter cells [4]. Once DNA gets condensed, a drastic decrease in RNA synthesis is observed [5]. Eventually, some cells can leave the cell cycle, temporarily (quiescent) or permanently (senescent), residing in a resting stage called G0, characterized by DNA content equal to G1 cells but minimal RNA levels. G0 cells are metabolically active: they perform their functions but are not ready for entering into the replication cycle [6,7]. A graphical representation of the overall process is given in Fig. 1a. Several methods are used to measure the cell cycle phase distribution. The most common one is the flow cytometry (FC) assessment of the DNA content per cell using propidium iodide (PI) staining, PI-FC. Despite the simplicity of the methodology, it can only distinguish among G0/G1, S and G2/M cells and additional
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Fig. 1. FTIRM assessment of the cell cycle phase–process flow. (a) Cartoon of cell growth and division through progressive cell-cycle stages: G1, S, G2 and M. Resting phase G0 is also represented; (b) DNA histogram retrieved from PI-FC analysis of a representative Asyn sample of the calibration dataset. The underlying distribution is recovered by fitting the G0/G1 (gray) and G2/M (blue) peaks as gaussian curves and the S phase (red) as a gaussian-broadened curve. In the inset, the cell-cycle phase distribution averaged on the triplicate Asyn experiments of calibration dataset is reported; (c) Hierarchical Cluster Analysis (HCA) of absorbance spectra based on Euclidean distances and Wards’ classification algorithm in the 1300–1000 cm−1 spectral region for the Asyn calibration dataset. In the inset, the percentages of the cells belonging to each cluster and its assignment are reported. Spectral centroids of each cluster ± standard deviations are plotted at the bottom of panel c. Area integral of the PhI band ± standard deviation is also reported for each cluster centroid. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
markers are necessary to further improve the classification. To distinguish G0 from G1 cells, RNA-binding dyes such as pyronin or acridin orange can be used, since it is known that cells in G0 have lower content of RNA compared to actively proliferating cells [8,9]. Similarly, to distinguish G2 from M cells, antibodies against phosphorylated histone H-3 [10] or mitotic phosphoprotein monoclonal antibody-2 (MPM-2) [11] can be exploited with FC analysis. Alternatively to FC measurements, immunohistochemical staining is also widely employed. As an example, Ki67 and mini-chromosome maintenance protein (MCM2) can be used to detect all proliferative cells, cyclins D and E to distinguish G1 stage, cyclin A or BrdU (bromodeoxyuridine) incorporation for Sphase discrimination and Cyclin B1 for G2 phase [12–14]. Novel markers and staining methods are being constantly developed, for example the use of fluorescent protein-p27 mutant fusion probe to distinguish G0 from G1 cells was recently reported [15]. In summary, a detailed classification of the cell cycle phases and their compartments is nowadays possible by using multiprobe analyses, which often require complex staining protocols and cell fixation. We recently demonstrated that Fourier Transform Infrared Microspectroscopy (FTIRM) in microfluidic devices is able to discriminate among G0/G1, S and G2/M phases of live B16 mouse melanoma cells with a degree of accuracy comparable with FC, without the use of labeling and staining protocols [16]. For the aim of the experiments presented in [16], B16 cell populations asynchronous (Asyn), synchronized in S phase by thymidine block (SynS) and synchronized in G0 by growing to confluence (SynG0), have been tested for cell cycle distribution by PI-FC. In parallel, microspectra of entire live B16 cells from each population have been collected in silicon-like BaF2 microfluidic devices with an optical path of 8 m, produced as described elsewhere [17]. For each FTIRM dataset, obtained by merging the results of triplicate experiments for both Asyn, SynS and SynG0 populations,
Hierarchical Cluster Analysis (HCA) of absorbance spectra based on Euclidean distances and Wards’ classification algorithm in the 1300–1000 cm−1 spectral region has been applied. In Fig. 1b–c, the workflow of the data analysis is exemplified for the collected Asyn dataset. The resulting dendrograms were cut according to the number of phases detected by PI-FC, namely three phases for Asyn populations (Asyn G0/G1, Asyn S, Asyn G2/M), two for SynS (SynS S, SynS G2/M) and one for SynG0 (G0/G1). The percentage of measured cells per cluster has been then calculated and each cluster tentatively assigned to a stage of the cell cycle according to the relative percentages obtained by PI-FC. The analysis of the spectral centroids revealed that the classification was mostly driven by the spectral intensity variations in the wavenumber range chosen for clustering. This interval is characterized by spectral contributions from nucleic acids (NAs), specifically the asymmetric (Phosphate I, PhI) and symmetric (Phosphate II, PhII) stretching bands of phosphodiester moieties of NAs’ backbone. PhI band has two major components, centered at ∼1240 (A-helical form of nucleic acids), and ∼1220 cm−1 (B-helical form of nucleic acids), respectively, and PhII is peaked at ∼1080 cm−1 . While the PhII band overlaps with several contributions coming from cellular sugars [18,19], their contribution to PhI band is less pronounced in mammalian cells. Stated the constancy of the optical path between different measurements, the spectral intensity of the PhI band (defined as the integrated area underneath the spectral region 1274–1182 cm−1 ) is proportional, to a first approximation, to the NA cellular content, as also reported by Whelan et al. [20]. The intensity of the PhI band for the analyzed datasets was the lowest for clusters assigned to G0/G1 in SynG0 and Asyn conditions, while it almost doubled for clusters assigned to G2/M stages in Asyn and SynS. For clusters assigned to S phases from Asyn and SynS synchronization it showed intermediate values. Summarizing, FTIRM of live B16 cells allows discriminating between different cell cycle phases on the basis of the solely cellular NA content
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determined as the area integral of PhI band, with a degree of accuracy comparable to FC with PI staining of cellular DNA. For the purpose of this work, a calibration dataset was built on the basis of the results already reported [16] and previously summarized, merging FTIRM data accordingly to the assigned cell cycle stage, and establishing the ranges of variability of the PhI area integral related to G0, G1, S and G2/M respectively (calibration intervals). The calibration intervals were used for assessing the cell cycle stage of each individual cell of a population of Asyn B16s (Asyn validation dataset) exclusively on the basis of the NA content, and the good accuracy of the classification was checked a posteriori by PI-FC. Moreover, we had a closer look into G0 and G1 phases, establishing that the continuous transition between quiescent and late G1 cells can be tracked by FTIRM, considering the gradual increase on RNA levels. To this aim, validation datasets were built, made of synchronized G0 and mitotic B16 cells: SynG0 and M validation datasets, respectively. Moreover, we pointed out that synchronization protocols can affect the cellular vibrational profile, by comparing the spectral profiles of mitotic cells derived by nocodazole synchronization with those released from G1-phase block and from cells normally progressing from an asynchronous population. Finally, in order to extend the applicability of the proposed methodology for cell cycle assessment of live mammalian cells by FTIRM on the basis of the NA content, we successfully performed a new set of experiments on human bone osteosarcoma cells, U2OS. 2. Materials and methods 2.1. Cell culture and synchronization B16 mouse melanoma cells were grown at 37 ◦ C in 5% CO2 in high-glucose Dulbecco’s modified Eagle’s medium (PAA) supplemented with 10% fetal bovine serum (Euroclone). B16 cell cycle lasts around 15–16 h: G1 (∼3 h), S (∼8 h), G2 (∼4 h), M (∼1 h) [21]. For more details on the synchronization protocols for SynS and SynG0 populations of the calibration dataset the reader is referred to [16]. For the synchronization (enrichment) in M-phase of validation dataset, cells were exposed to 2 mM thymidine (Sigma) for 16 h, released for 9 h, exposed to 0.1 g/mL nocodazole (Sigma) for 16 h and collected. A second approach was also tested: cells after a thymidine block were released for 9 h, exposed for 16 h to 5 g/mL aphidicolin and released for 6 h. To obtain SynG0 population of the validation dataset, cells were grown to confluence for 4 days without adding fresh media. Before measurements, B16 cells were collected by trypsinization, washed in PBS and split to two parts for the parallel analysis by FTIRM and flow cytometry (FC). U2OS human bone osteosarcoma cells were grown at 37 ◦ C in 5% CO2 in high-glucose Dulbecco’s modified Eagle’s medium (PAA) supplemented with 10% fetal bovine serum (Euroclone). The approximate time for U2OS cell to divide is about 30 h [22]. Only asynchronous populations have been considered for the aims of this paper. Before measurements, cells were collected by trypsinization, washed in PBS and split to two parts for the parallel analysis by FTIRM and flow cytometry (FC). 2.2. Flow-cytometry Measurement of DNA content by FC was exploited to assess cell cycle phase distribution of both B16 and U2OS cells. Once trypsinized, cells were resuspended in PBS and fixed with ethanol at a final concentration of 70% and minimum 1 h incubation at 4 ◦ C. Cells were washed and resuspended in PBS containing 1% FBS. DNA was stained for 3 h with 50 g/mL propidium iodide (PI) and RNA removed by treatment with 200 g/mL RNaseA.
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FC analysis was performed using FACSCalibur (Becton Dickinson) and the cell cycle phase distribution was calculated with the CellQuestPro and Modfit LT 3.0 software. 2.3. Ki67 staining To determine the percentage of non-proliferating (G0) B16 cells, Ki67 staining was performed. Asynchronous (Asyn calibration dataset) or G0-synchronized (both SynG0 calibration and validation datasets) cells were plated on coverslips (5000 cells per slide in a 100 L drop) and left to attach for 20 min. Cells were then fixed in 3.7% paraformaldehyde, blocked in 4% BSA and incubated in anti Ki67 primary antibody (Novus Biologicals, 1:50 in 1% BSA in PBS), followed by an incubation in Alexa488 anti-rabbit (Invitrogen, Molecular Probes, 1:300). Nuclei were counterstained with Toto3 (Invitrogen Molecular Probes, 1:5000). Slides were mounted in Vectashield mounting media (Vector labs), sealed and analyzed under Zeiss LSM 510 Meta confocal microscope. Images were acquired using the LSM software and the percentage of cells in G0 phase was calculated from at least 200 cells as the number of Ki67 negative cells versus all cells stained by Toto3. 2.4. FTIRM data collection Upon trypsinization, B16 mouse melanoma cells or U2OS human osteosarcoma cells were resuspended in NaCl 0.9% physiological solution, to a final concentration of approximately 1 × 106 cells/mL. Microfluidic devices were obtained by UV-lithography on 0.5 mm thick CaF2 optical windows. For all of them, the optical path was set to 8 m. Details on the fluidic chip geometries and on the fabrication protocols used are described elsewhere [17,23]. Note that both B16 and U2OS cells have been measured immediately after trypsinization for ∼2 h, without allowing them to adhere onto the substrate. Under these conditions, both cell types are spheroids, the average diameter of which is in the range of 10–15 m, slightly bigger that the optical path constrained by the fluidic cell. Therefore, contributions to spectral intensity modulation from possible cellular thickness variations during cell cycle progression are minimal. IR microspectra of individual cells were acquired at SISSI beamline [24] at Elettra – Sincrotrone Trieste, which is equipped with a Vis-IR microscope Hyperion 3000 coupled to a Vertex70 interferometer (Bruker Optics GmbH, Ettlingen, Germany). Microscope knife-edge apertures were set in order to fit the size of each cell (from 15 × 15 to 40 × 40 m). A Mercury-CadmiumTelluride detector with a 100 m sensitive element was used. 256 scans were averaged for each measurement in the wavenumber region 6000–800 cm−1 in transmission mode using a 15× condenser/objective at a spectral resolution of 4 cm−1 . BlackmanHarris 3-term apodization function and filling factor of 2 were chosen. For each cell measured, a buffer point close to the cell was also collected. Both cell and buffer spectra were rationed against air background. Each measurement run lasted no more than two hours in order to guarantee cell synchronization and avoid spectral artifacts derived from buffer waste. For the purpose of the paper, it has to be highlighted that the calibration dataset has been measured several months before the validation experiments, using silicon-like BaF2 devices (1 mm + 1 mm window thickness, 8 m optical path) [17], while for the validation datasets devices made on raw CaF2 (0.5 + 0.5 mm windows thickness, 8 m optical path) were used. This was done in order to verify the possible effects on the intensity of the phosphate bands due to the lower transparency of CaF2 substrates below 1100 cm−1 with respect to BaF2 . The consistency among calibration and validation data allows concluding that the different transmittance of these substrates has negligible effects on the reliability
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and reproducibility of the results, stated the comparable refractive index among the two [25].
2.5. FTIRM data analysis Water medium spectral contribution was subtracted from each raw cell microspectrum by using the in-house optimized Matlab routine described by Vaccari et al. [26]. Before spectral subtraction, the atmospheric compensation and offset correction routine of OPUS 6.5 (Bruker Optics GmbH, Ettlingen, Germany) were applied. Spectra from the B16 calibration dataset (SynG0, SynS and Asyn) were independently analyzed using a multivariate approach in R environment, HyperSpec [27,28] and stats packages. Briefly, Hierarchical Cluster Analysis (HCA) was performed on the absorbance spectra, based on Euclidean distances and Ward’s classification algorithm, in the 1300–1000 cm−1 spectral region. The number of clusters was chosen by cutting off the dendrograms according to the level of heterogeneity and the number of cell cycle phases identified by FC. The assignment of each cluster to a specific phase of the cell cycle was done cross-checking the consistency of the percentages among FC and FTIRM data. The reader is referred to [16] for more details. The same procedure was applied for the samples of U2OS human osteosarcoma cells. Calibration intervals for both cell lines have been then calculated as the average ± standard deviation of the area integral of the PhI band (1274–1182 cm−1 ) of all the files belonging to the same stage of the cell cycle, as assessed by FTIRM. For the B16 validation datasets, the area integral of the phosphate asymmetric stretching bands (PhI, 1274–1182 cm−1 ) was computed using OPUS 6.5 software. FTIRM histograms of the distribution of the PhI band intensity were calculated with Origin 8.1 with a binning of 0.02 a.u. and the cell cycle phase distribution assessed on the basis of the calibration intervals. One-way analysis of variance, ANOVA, has been performed with Origin 8.1. For the evaluation of the differences among the spectral profiles of mitotic cells, second derivatives of the subtracted spectra were computed by applying Savitzky–Golay algorithm (13 smoothing points) and vector normalized (OPUS 6.5 software). HCA on second derivatives has been performed in the 1200–950 cm−1 spectral region (Euclidean distances and Ward’s classification algorithm) using HyperSpec [27,28] and stats packages in R environment.
3. Results and discussion
Therefore we tentatively linked the difference in the total NA content to the diverse RNA content between G0 and G1 phases [3,29]. In order to confirm this hypothesis, B16 mouse melanoma cells from SynG0 and Asyn of calibration datasets have been immunostained for Ki67 protein, for evaluating the percentage of G0-quiescent cells. To this aim, at least 200 cells were counted for each set, an amount comparable with the cells sampled by FTIRM. Ki67 protein is a cellular marker for proliferation, expressed during all progressive phases of the cell cycle, but absent for cells in G0. Immunostaining combined with FC assessment of PI-stained DNA revealed that almost 90% of the entire population of SynG0 was induced to quiescence, while only a small fraction of the Asyn one (∼30%) was resting. An example of the Ki67 immunostaining is shown in Fig. 2, where the results for Asyn and a SynG0 are compared. Since SynG0 calibration dataset is almost entirely constituted by cells in G0 phase, in this analysis it is still considered as a unique cluster. On the contrary, the dendrogram for Asyn calibration dataset, shown in Fig. 1b, was further cut, and we could distinguish two sub-clusters of Asyn G0/G1: the larger one (∼60% of the G0/G1 population) was tentatively assigned to G1 phase (Asyn G1), the smaller (∼40% of the G0/G1 populations) to G0 (Asyn G0). The consistency of the assignment was cross-checked by calculating the average intensity of the area integral of the PhI band for the spectra belonging to the two clusters, that was lower for Asyn G0 than for Asyn G1, confirming our hypothesis. In the end, all spectra assigned to the same phase of the cell cycle (as established by FTIRM of live B16 cells: G0, G1, S and G2/M), have been merged, and the average value of the PhI band intensity has been calculated. The results are summarized in Table 1 and show that differences in NA content among the groups were statistically significant (one-way analysis of variance, ANOVA, p = 0.05). Ranges of variability of the PhI band area integral can be identified for G0, G1, S and G2/M phases, defined as the mean value for each stage minus (Min value) and plus (Max value) standard deviations, and they are potentially useful as calibration intervals to estimate the belonging of an individual B16 cell to a cell cycle stage, exclusively on the basis of its NA content. In order to validate this hypothesis, three different samples have been measured by FTIRM in CaF2 -based devices 8-m thick: B16 cells asynchronous (Asyn validation dataset from now on), B16 cells synchronized in G0 phase by growing to confluence (SynG0 validation dataset from now on) and mitotic cells (M validation dataset hereon). The distribution of the cell cycle stages for each validation dataset was then established exploiting Calibration Intervals and the classification verified a posteriori by PI-FC and, for SynG0 validation dataset, also by immunohistochemistry using Ki67 staining.
3.1. Methodology validation The first objective of this paper is to demonstrate that the cell cycle phase of an individual live cell can be assessed by FTIRM on the base of its NA content, provided that the ranges of variability of the PhI band related to each stage of the cellular progression are defined. To this aim, we first refined the data analysis of the set of data reported in [16], named calibration dataset hereafter. In that paper, we pointed out that the mean NA content for SynG0 G0/G1, established on the basis of the area integral of the PhI band (1274–1182 cm−1 ), was almost two-thirds of the one established for the same stage of asynchronous population (Asyn G0/G1), 0.210 ± 0.035 and 0.309 ± 0.050 respectively, possibly due to a different ratio between G1 and G0 cells in the two samples. Indeed, a higher percentage of quiescent cells upon growth to confluence is expected [3,29]. The RNA content of G0 cells is lower than in any other progressive stage of the cell cycle and it is almost half of that in G1 [30]. The level of ribosomal RNA is particularly low because the protein translation is less active [31].
3.1.1. Asyn validation dataset FTIRM of live asynchronous B16 cells, Asyn validation dataset, has been performed as described in Section 2. According to the area integral of the PhI band, it was established that 29% of the measured cells reside in the G0 calibration interval, 31% in the G1, 28% and 12% in the S and G2/M, respectively. Fig. 3c shows the recurrence of PhI integral area values for all the cells of the Asyn validation dataset. PI-FC gave the following output: G0/G1 53.4%; S 35.1%; G2/M 11.5% (see Fig. 3a). FTIRM and FC results are in good agreement. 3.1.2. SynG0 and M validation datasets FTIRM analysis of SynG0 B16 cells of the validation dataset allowed to determine that 51% of the cell were proliferating (35% G1, 15% S, 1% G2/M), while 49% lasted in the quiescent G0 phase (see Fig. 3c). From FC, it was possible to establish that a small percentage of cells were in S (∼9%) and G2/M phase (∼8%) upon synchronization by growth to confluence and all other (∼83%) were in G0/G1 (see Fig. 3b). Combining immunostaining for Ki67, we determined
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Fig. 2. Identification of B16 non-proliferating cells by immunostaining for Ki67. (a, d) Ki67 stained in green for SynG0 and Asyn cells; (b, e) nuclei stained with Toto3 in purple for SynG0 and Asyn cells; (c, f) merge of Ki67 and Toto3 staining for SynG0 and Asyn cells (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
that ∼77% of the cells were in G0. FTIRM data, plotted in Fig. 3d, reveal enrichment in the G0 phase with respect to the progressive phases of the cell cycle when compared to Asyn validation dataset (see Fig. 3c), while it was not possible to precisely reproduce the proportion between G1 and G0 phases obtained with Ki67 staining. Indeed, the transition from G0 to G1 is smooth, without a clear border among the two stages in terms of total NA content, but it is characterized by an intermediate population of post-mitotic cells that are increasing their RNA concentration above the restriction point for DNA replication, the early G1 cells. Those cells should have a total NA content in between G0 and late G1 cells. For verifying this point, a M validation dataset was built by enriching a B16 population in the mitotic phase using nocodazole, as described in Section 2. The enrichment of the M-phase was only ∼20% (see Fig. 4a), but it was enough for allowing an easier visualization and faster selection of mitotic cells based on morphology. As a matter of fact, for the purpose of the experiment, only cells characterized by membrane invagination, possibly in the stage of mitosis before cytokinesis,
were selected (see examples in Fig. 5, inset) and measured. For this sample, a histogram of the distribution of the PhI band was also generated and it is shown Fig. 4b. Almost all selected cells fall in the G2/M calibration interval, and all of them are represented by the Gaussian distribution of the PhI band for G2/M calibration dataset. On the background of Fig. 4b, the recurrence of the PhI of the G2/M calibration dataset is plotted in light gray, in order to better appreciate this behavior. The average value of the PhI band area integral for these cells was of 0.595 ± 0.061 a.u.. It is known that, when cells divide, daughter cells become two early G1 cells [3], and it is therefore not surprising that half of the area integral value of the PhI band for the M validation dataset is 0.297 ± 0.061 a.u., that is located in between G0 and late G1. Summarizing, the validation experiments here reported demonstrate that the cell-cycle phase distribution of B16 cells can be defined using FTIRM, only on the basis of the PhI area integral once established a calibration dataset, with a degree of accuracy comparable to FC. Moreover, the enrichment of a cell population in G0 can
Table 1 In column three (average value ± sd (a.u.)), the average values of the PhI band area integral obtained merging all the spectra of each cluster of the calibration dataset (second column, clusters) assigned to the same phase of the cell cycle (first column, phase) are reported. Differences in the NA content among the phases were statistically significant for p = 0.05 (one-way analysis of variance, ANOVA). Calibration Intervals, calculated as the average value of each phase plus/minus standard deviation are reported in the fourth column. Calibration intervals Phase
Clusters
G0
Asyn G0 Syn G0 Asyn G1 Asyn S SynS S Asyn G2/M SynS G2/M
G1 S G2/M
Average value ± SD (a.u.)
Calibration interval (a.u.)
0.233 ± 0.066
0.167–0.300
0.354 ± 0.054 0.479 ± 0.074
0.300–0.408 0.408–0.549
0.676 ± 0.127
0.549–0.803
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Fig. 3. Asyn and SynG0 Validation dataset for B16 cells – recurrence of PhI band. (a) DNA histogram and cell cycle distribution retrieved from PI-FC of the Asyn validation dataset. The underlying distribution is recovered by fitting the G0/G1 (gray) and G2/M (blue) peaks as Gaussian curves and the S phase (red) as a Gaussian-broadened curve; (b) DNA histogram and cell cycle distribution retrieved from PI-FC of the SynG0 validation dataset. The underlying distribution is recovered as in (a); (c) Recurrence of PhI integral area values for Asyn validation dataset and cell-cycle distribution evaluated according to calibration intervals. Histogram bars (binning 0.05 a.u.) in G0 calibration interval are black, in G1 green, in S red and in G2/M blue; (d) Recurrence of PhI integral area values for SynG0 validation dataset and cell-cycle distribution evaluated according to calibration intervals. The same color code of (d) is used. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
also be established, while the assessment of G0 versus G1 percentages with a degree of accuracy comparable to PI-FC assisted by Ki67 staining is hindered by the smooth increase of RNA levels between quiescent and progressive stages. 3.2. Effect of nocodazole The main advantage in using FTIRM as an alternative technique for the determination of the cell cycle phase distribution is the possibility to perform an in situ sorting of live cells without the need of labeling, staining or fixation of the sample. FTIRM indeed does
not affect the viability of the cells and moreover provides additional information on the biochemical content of living cells apart from nucleic acids. We have recently demonstrated that fixation protocols may alter the biochemical content and spectral profile of a cell, in particular in the nucleic acids region [26]. Moreover, there are several papers that report and/or warn on the possible adverse effects of synchronization protocols on cellular biochemistry [32,33]. Therefore, the availability of a label-free technique for the in vitro determination of the cellular phase could benefit modern cellular biology. As an example, we investigated the effects of synchronization protocols on cellular biochemistry by comparing
Fig. 4. M Validation dataset for B16 cells (a) DNA histogram and cell cycle distribution retrieved from PI-FC of the M validation dataset. The underlying distribution is recovered by fitting the G0/G1 (gray) and G2/M (blue) peaks as Gaussian curves and the S phase (red) as a Gaussian-broadened curve; (b) Recurrence of PhI integral area values for mitotic cells of M validation dataset. Histogram bars (binning 0.05 a.u.) in S calibration interval are in red and in G2/M in blue. On the background, the recurrence of the PhI of the G2/M calibration dataset is plotted in light gray. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 5. Effects of the different synchronization protocols in M phase (a) HCA performed on vector normalized second derivatives of mitotic cells coming from three different preparations (i) synchronization by nocodazole, (ii) synchronization by G1 block (using thymidine and aphidicolin) and release to M-phase, and (iii) mitotic cells from an asynchronous population. Two main clusters are obtained, one made by nocodazole treated cells and the other by mitotic cells from normally progressive populations. The inset shows the type of cells chosen for the measurements by morphologycriteria; (b) cluster centroids related to nocodazole treated cells (red) and the other two preparations (blue). Line thickness is proportional to the standard deviation of each centroid (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
spectra of mitotic live cells following three different approaches: (i) synchronization by nocodazole, (ii) synchronization by G1 block (using thymidine and aphidicolin) and release to M-phase, and (iii) mitotic cells from an asynchronous population. Cell spectra have been selected according to the shape criteria defined in Section 3.2. Fig. 5a reports the results of HCA analysis performed on the second derivatives of the subtracted spectra for the three M groups in the 1200–950 cm−1 spectral region. Despite the low spectral heterogeneity levels, two main clusters are detected: the first one constituted of mitotic cells treated with nocodazole, the second of mitotic cells from G1block and asynchronous populations. Cluster centroids are shown in Fig. 5b. Some specific differences for the enrichment with nocodazole are detected. In particular, a displacement from 1049 to 1053 cm−1 and from 1014 to 1025 cm−1 of the C O stretching coupled with C O bending of C OH moieties of cellular carbohydrates (see Fig. 5b) [34–36]. Moreover, the variation of the spectral shape in the range 1165–1145 cm−1 could be possibly assigned to C O stretching of C OH groups [37] and therefore related to variations in cellular carbohydrates. All these spectral variations could be tentatively correlated to the mode of action of nocodazole. Nocodazole blocks the progression of mitosis by inhibiting the formation of microtubules [38] and it has also been reported to disturb the transport of newly synthesized proteins [39]. Moreover, it has been found that arresting cells in G2/M using anti-microtubule agents (such as nocodazole) causes a
noticeable increase in the concentration of N-acetylglucosamine (GlcNAc) residues on numerous glycoproteins [40]. This altered glycosilation was found to be independent from microtubule disassembly and not directly related to G2/M phase of the cell cycle [41]. This is in agreement with the observation that normally progressing cells (Asyn G2/M and M release from G1) cluster together, while changes in glycosilation may be responsible for the shifts observed in the spectral contributions of cellular sugars in the 1200–950 cm−1 spectral region, as also reported by other authors [42]. 3.3. Assessment of the cell-cycle distribution of U2OS cell line by FTIRM The possibility to discriminate among different stages of the cell cycle by FTIRM on the basis of the NA content might have a general applicability for mammalian cells. Indeed, despite the specific genomic profile of each cell line, the relative variations of cellular DNA and RNA follow comparable trends during cellular progression. In order to demonstrate that the proposed methodology could be extended to other cell lines, it was applied to analyze the phase cycle distribution of U2OS human bone osteosarcoma cells. Specifically, we built a test dataset for U2OS cells following a simplified approach with respect to the one implemented in [16], and summarized in the introduction. Only asynchronous
Fig. 6. Calibration dataset for U2OS. (a) DNA histogram retrieved from PI-FC analysis of a representative Asyn sample of the U2OS test dataset. The underlying distribution is recovered by fitting the G0/G1 (gray) and G2/M (blue) peaks as gaussian curves and the S phase (red) as a gaussian-broadened curve; (b) Hierarchical Cluster Analysis (HCA) of absorbance spectra based on Euclidean distances and Wards’ classification algorithm in the 1300–1000 cm−1 spectral region for the U2OS Asyn test dataset. In the inset, the percentages of the cells belonging to each cluster and the cluster assignment are reported; (c) Plot of the spectral centroids of each cluster ± standard deviation (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Table 2 Cell cycle phase distribution obtained for U2OS human osteosarcoma cells using FC with PI staining averaged on a triplicate experiment is reported in column “FC results”; cell cycle phase distribution obtained by Hierarchical Cluster Analysis (HCA) of absorbance spectra based on Euclidean distances and Wards’ classification algorithm in the 1300–1000 cm−1 spectral region for the same U2OS cell population is reported in column “HCA results”. Area integral of the PhI band ± standard deviation is also reported for each cluster centroid in column “Average value ± sd (a.u.)”. Differences in the NA content among the phases were statistically significant for p = 0.05 (one-way analysis of variance, ANOVA). Calibration Intervals, calculated as the average value of each phase plus/minus standard deviation are also reported in the last column (Calibration Interval (a.u.)). Phase
FC results (%)
HCA results (%)
Average value ± sd (a.u.)
Calibration interval (a.u.)
G0/G1 S G2/M
41.5 ± 12.0 19.5 ± 0.7 39 ± 12.7
45.90 23.50 30.60
0.181 ± 0.044 0.264 ± 0.043 0.324 ± 0.064
0.137–0.226 0.220–0.307 0.256–0.389
populations of U2OS cells have been taken into account, in order to make the protocol faster. Parallel FTIRM and PI-FC experiments on an asynchronous population of U2OS cells has been done, and repeated in triplicate. PI-FC analysis allowed to establish the following distribution: G0/G1 41.5 ± 12.0%, S 19.5 ± 0.7%, G2/M 39.0 ± 12.7% (standard deviations are related to the triplicate). Fig. 6a shows the FC histogram for one of the Asyn U2OS samples. HCA on absorbance microspectra of the entire test dataset in the 1300–1000 cm−1 spectral region provided the dendrogram shown in Fig. 6b. The dendrogram was cut into three clusters, according to FC results. Clusters were then assigned to the G0/G1, S and G2/M phases conforming to the relative percentages. 45.9% of the cell population was classified in the G0/G1 phase by FTIRM, 23.50% in S and 30.6% in G2/M. The correctness of the assignment was checked by retrieving the average NA content per each cluster, defined as the average of the area integral of the PhI band for all the microspectra belonging to it (see Fig. 6c). In agreement with the results obtained for B16 cells, the intensity of this band almost doubled from G0/G1 to G2/M (see Table 2), confirming the predictive power of the proposed methodology also for U2OS cells and the comparable accuracy of FTIRM and PI-FC.
4. Conclusions In this work, we showed that, once built a calibration dataset for assessing the cell cycle stages of live B16 melanoma cells and defined calibration intervals based on the average intensity of the PhI band, these can be exploited in subsequent experiments for determining the status of any individual live B16 cell, with a degree of accuracy comparable to FC determination with PI staining of G0/G1, S and G2/M phases. In addition, it was possible to verify that FTIRM may discriminate to some extent between G0 and late G1 cells. These results might have a general value for mammalian cell line, as proved for U2OS human bone osteosarcoma cells. FTIRM can be performed on live cells, which can be therefore sorted within the fluidic device according to their progression state. Those cells can be then tested for their individual response to chemical–thermal–mechanical stresses, being able to assert the response specificity linked to the cell-cycle stage, following a totally label-free experimental approach. The FTIRM-based method here proposed does not require the enrichment of the cell population to a specific cell-cycle stage through synchronization protocols. Indeed, synchronization protocols may induce biochemical changes, and consequent spectral variations, not directly related to the specific cell-cycle stage, but to the procedures followed for population enrichment. Actually, specific changes induced by nocodazole in B16s were found, when comparing to mitotic cells for normally progressive populations. Therefore, the possibility to simply identify the cell cycle phase of an individual cell on the basis of its NA content by FTIRM is of paramount importance and offers new perspectives in many fields of science where this knowledge is fundamental.
Acknowledgements This study was partially financed from the Italy–Slovenia CrossBorder Cooperation Programme, 2007–2013, project Glioma. The authors thank Dr. Alessandro Marcello for support on flow cytometric measurements.
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Contents lists available at ScienceDirect
Vibrational Spectroscopy journal homepage: www.elsevier.com/locate/vibspec
Further insights into the assessment of cell cycle phases by FTIR microspectroscopy Diana E. Bedolla a , Saˇsa Kenig a , Elisa Mitri b , Paola Storici a , Lisa Vaccari a,∗ a b
Elettra – Sincrotrone Trieste, S.S. 14 Km 163.5, 34149 Basovizza, Trieste, Italy CNR-IOM, TASC Laboratory, S.S. 14 Km 163.5, 34149 Basovizza, Trieste, Italy
a r t i c l e
i n f o
Article history: Available online 1 September 2014 Keywords: FTIR microspectroscopy Flow cytometry Cell cycle B16 mouse melanoma cells U2OS human bone osteosarcoma cells
a b s t r a c t Cell growth and replication occurs in an orderly manner through a set of tightly coordinated physiological events, classified as G0, G1, S, G2 and M in conformity to their characteristics. In a previous work, by combining the results of flow cytometry (FC) using propidium iodide (PI) staining, PI-FC, and Fourier Transform Infrared Microspectroscopy (FTIRM), we gathered information to classify live B16 cells into three different set of phases (G0/G1, S and G2/M), according to their nucleic acid content measured as the area integral of the Phosphate I band (PhI, 1274–1182 cm−1 ). In this work, we demonstrate that, once built a calibration dataset for a cell line determining the intervals of the PhI area integral related to each phase of the cell cycle, such data can be used for assigning the stage to which a live cell belongs without the support of FC. In addition, we evaluate the spectral profile of early G1 B16 cells, and compare it with the one of G0 and late G1 cell cycle phases. FTIRM highlights that G0 and G1 phases are a continuum, where the content of RNA of early G1 cells is in between G0 and late G1, and the overall nucleic acid content varies accordingly. In the paper, we also pinpoint the effects on synchronization protocols on cellular biochemistry, further strengthening the potentialities of a totally label-free methodology for cell sorting. Finally, we demonstrate that the general concept behind the proposed approach may be extended to other mammalian cell lines: human bone osteosarcoma (U2OS) cells were tested. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Eukaryotic cells follow an ordered set of growth and division events known as cell cycle [1]. The cell cycle is divided into four main steps, called Gap 1 (G1), Synthesis (S), Gap 2 (G2) and Mitosis (M), each of them characterized by peculiar cellular activities [2]. The G1 phase, also called growth phase, starts right after the mitotic process and is characterized by protein and mRNA synthesis as well as by production of more cell organelles; the volume of the cytoplasm increases and the cell size follows the same trend. G1 stage is further divided into early G1 and late G1, sometimes also called G1A and G1B [3]. Cells in late G1 have higher content of both RNA and proteins compared to post-mitotic early G1 cells, and can directly enter the S phase, while cells in early G1 need to increase their RNA concentration above a threshold level to be able to initiate DNA replication (restriction point). In the following S phase,
Abbreviations: FTIRM, Fourier Transform Infrared Microspectroscopy; FC, flow cytometry; PI, propidium iodine; NA, nucleic acid. ∗ Corresponding author. Tel.: +39 040 3758567; fax: +39 040 9380902. E-mail address: [email protected] (L. Vaccari). http://dx.doi.org/10.1016/j.vibspec.2014.08.007 0924-2031/© 2014 Elsevier B.V. All rights reserved.
the cell commits to DNA replication. At the end of the S phase, the amount of DNA in the cell is doubled and two identical set of chromosomes are produced. The G2 phase lasts until the cell enters in mitosis and it is characterized by unaffected DNA content while an intense bio-synthetic activity takes place. In five consecutive stages of M phase (prophase, prometaphase, metaphase, anaphase, telophase/cytokinesis) nuclear membrane disintegrates, DNA condenses and chromosomes are separated and split into two equal portions. In the end, the cell divides into two identical daughter cells [4]. Once DNA gets condensed, a drastic decrease in RNA synthesis is observed [5]. Eventually, some cells can leave the cell cycle, temporarily (quiescent) or permanently (senescent), residing in a resting stage called G0, characterized by DNA content equal to G1 cells but minimal RNA levels. G0 cells are metabolically active: they perform their functions but are not ready for entering into the replication cycle [6,7]. A graphical representation of the overall process is given in Fig. 1a. Several methods are used to measure the cell cycle phase distribution. The most common one is the flow cytometry (FC) assessment of the DNA content per cell using propidium iodide (PI) staining, PI-FC. Despite the simplicity of the methodology, it can only distinguish among G0/G1, S and G2/M cells and additional
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Fig. 1. FTIRM assessment of the cell cycle phase–process flow. (a) Cartoon of cell growth and division through progressive cell-cycle stages: G1, S, G2 and M. Resting phase G0 is also represented; (b) DNA histogram retrieved from PI-FC analysis of a representative Asyn sample of the calibration dataset. The underlying distribution is recovered by fitting the G0/G1 (gray) and G2/M (blue) peaks as gaussian curves and the S phase (red) as a gaussian-broadened curve. In the inset, the cell-cycle phase distribution averaged on the triplicate Asyn experiments of calibration dataset is reported; (c) Hierarchical Cluster Analysis (HCA) of absorbance spectra based on Euclidean distances and Wards’ classification algorithm in the 1300–1000 cm−1 spectral region for the Asyn calibration dataset. In the inset, the percentages of the cells belonging to each cluster and its assignment are reported. Spectral centroids of each cluster ± standard deviations are plotted at the bottom of panel c. Area integral of the PhI band ± standard deviation is also reported for each cluster centroid. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
markers are necessary to further improve the classification. To distinguish G0 from G1 cells, RNA-binding dyes such as pyronin or acridin orange can be used, since it is known that cells in G0 have lower content of RNA compared to actively proliferating cells [8,9]. Similarly, to distinguish G2 from M cells, antibodies against phosphorylated histone H-3 [10] or mitotic phosphoprotein monoclonal antibody-2 (MPM-2) [11] can be exploited with FC analysis. Alternatively to FC measurements, immunohistochemical staining is also widely employed. As an example, Ki67 and mini-chromosome maintenance protein (MCM2) can be used to detect all proliferative cells, cyclins D and E to distinguish G1 stage, cyclin A or BrdU (bromodeoxyuridine) incorporation for Sphase discrimination and Cyclin B1 for G2 phase [12–14]. Novel markers and staining methods are being constantly developed, for example the use of fluorescent protein-p27 mutant fusion probe to distinguish G0 from G1 cells was recently reported [15]. In summary, a detailed classification of the cell cycle phases and their compartments is nowadays possible by using multiprobe analyses, which often require complex staining protocols and cell fixation. We recently demonstrated that Fourier Transform Infrared Microspectroscopy (FTIRM) in microfluidic devices is able to discriminate among G0/G1, S and G2/M phases of live B16 mouse melanoma cells with a degree of accuracy comparable with FC, without the use of labeling and staining protocols [16]. For the aim of the experiments presented in [16], B16 cell populations asynchronous (Asyn), synchronized in S phase by thymidine block (SynS) and synchronized in G0 by growing to confluence (SynG0), have been tested for cell cycle distribution by PI-FC. In parallel, microspectra of entire live B16 cells from each population have been collected in silicon-like BaF2 microfluidic devices with an optical path of 8 m, produced as described elsewhere [17]. For each FTIRM dataset, obtained by merging the results of triplicate experiments for both Asyn, SynS and SynG0 populations,
Hierarchical Cluster Analysis (HCA) of absorbance spectra based on Euclidean distances and Wards’ classification algorithm in the 1300–1000 cm−1 spectral region has been applied. In Fig. 1b–c, the workflow of the data analysis is exemplified for the collected Asyn dataset. The resulting dendrograms were cut according to the number of phases detected by PI-FC, namely three phases for Asyn populations (Asyn G0/G1, Asyn S, Asyn G2/M), two for SynS (SynS S, SynS G2/M) and one for SynG0 (G0/G1). The percentage of measured cells per cluster has been then calculated and each cluster tentatively assigned to a stage of the cell cycle according to the relative percentages obtained by PI-FC. The analysis of the spectral centroids revealed that the classification was mostly driven by the spectral intensity variations in the wavenumber range chosen for clustering. This interval is characterized by spectral contributions from nucleic acids (NAs), specifically the asymmetric (Phosphate I, PhI) and symmetric (Phosphate II, PhII) stretching bands of phosphodiester moieties of NAs’ backbone. PhI band has two major components, centered at ∼1240 (A-helical form of nucleic acids), and ∼1220 cm−1 (B-helical form of nucleic acids), respectively, and PhII is peaked at ∼1080 cm−1 . While the PhII band overlaps with several contributions coming from cellular sugars [18,19], their contribution to PhI band is less pronounced in mammalian cells. Stated the constancy of the optical path between different measurements, the spectral intensity of the PhI band (defined as the integrated area underneath the spectral region 1274–1182 cm−1 ) is proportional, to a first approximation, to the NA cellular content, as also reported by Whelan et al. [20]. The intensity of the PhI band for the analyzed datasets was the lowest for clusters assigned to G0/G1 in SynG0 and Asyn conditions, while it almost doubled for clusters assigned to G2/M stages in Asyn and SynS. For clusters assigned to S phases from Asyn and SynS synchronization it showed intermediate values. Summarizing, FTIRM of live B16 cells allows discriminating between different cell cycle phases on the basis of the solely cellular NA content
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determined as the area integral of PhI band, with a degree of accuracy comparable to FC with PI staining of cellular DNA. For the purpose of this work, a calibration dataset was built on the basis of the results already reported [16] and previously summarized, merging FTIRM data accordingly to the assigned cell cycle stage, and establishing the ranges of variability of the PhI area integral related to G0, G1, S and G2/M respectively (calibration intervals). The calibration intervals were used for assessing the cell cycle stage of each individual cell of a population of Asyn B16s (Asyn validation dataset) exclusively on the basis of the NA content, and the good accuracy of the classification was checked a posteriori by PI-FC. Moreover, we had a closer look into G0 and G1 phases, establishing that the continuous transition between quiescent and late G1 cells can be tracked by FTIRM, considering the gradual increase on RNA levels. To this aim, validation datasets were built, made of synchronized G0 and mitotic B16 cells: SynG0 and M validation datasets, respectively. Moreover, we pointed out that synchronization protocols can affect the cellular vibrational profile, by comparing the spectral profiles of mitotic cells derived by nocodazole synchronization with those released from G1-phase block and from cells normally progressing from an asynchronous population. Finally, in order to extend the applicability of the proposed methodology for cell cycle assessment of live mammalian cells by FTIRM on the basis of the NA content, we successfully performed a new set of experiments on human bone osteosarcoma cells, U2OS. 2. Materials and methods 2.1. Cell culture and synchronization B16 mouse melanoma cells were grown at 37 ◦ C in 5% CO2 in high-glucose Dulbecco’s modified Eagle’s medium (PAA) supplemented with 10% fetal bovine serum (Euroclone). B16 cell cycle lasts around 15–16 h: G1 (∼3 h), S (∼8 h), G2 (∼4 h), M (∼1 h) [21]. For more details on the synchronization protocols for SynS and SynG0 populations of the calibration dataset the reader is referred to [16]. For the synchronization (enrichment) in M-phase of validation dataset, cells were exposed to 2 mM thymidine (Sigma) for 16 h, released for 9 h, exposed to 0.1 g/mL nocodazole (Sigma) for 16 h and collected. A second approach was also tested: cells after a thymidine block were released for 9 h, exposed for 16 h to 5 g/mL aphidicolin and released for 6 h. To obtain SynG0 population of the validation dataset, cells were grown to confluence for 4 days without adding fresh media. Before measurements, B16 cells were collected by trypsinization, washed in PBS and split to two parts for the parallel analysis by FTIRM and flow cytometry (FC). U2OS human bone osteosarcoma cells were grown at 37 ◦ C in 5% CO2 in high-glucose Dulbecco’s modified Eagle’s medium (PAA) supplemented with 10% fetal bovine serum (Euroclone). The approximate time for U2OS cell to divide is about 30 h [22]. Only asynchronous populations have been considered for the aims of this paper. Before measurements, cells were collected by trypsinization, washed in PBS and split to two parts for the parallel analysis by FTIRM and flow cytometry (FC). 2.2. Flow-cytometry Measurement of DNA content by FC was exploited to assess cell cycle phase distribution of both B16 and U2OS cells. Once trypsinized, cells were resuspended in PBS and fixed with ethanol at a final concentration of 70% and minimum 1 h incubation at 4 ◦ C. Cells were washed and resuspended in PBS containing 1% FBS. DNA was stained for 3 h with 50 g/mL propidium iodide (PI) and RNA removed by treatment with 200 g/mL RNaseA.
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FC analysis was performed using FACSCalibur (Becton Dickinson) and the cell cycle phase distribution was calculated with the CellQuestPro and Modfit LT 3.0 software. 2.3. Ki67 staining To determine the percentage of non-proliferating (G0) B16 cells, Ki67 staining was performed. Asynchronous (Asyn calibration dataset) or G0-synchronized (both SynG0 calibration and validation datasets) cells were plated on coverslips (5000 cells per slide in a 100 L drop) and left to attach for 20 min. Cells were then fixed in 3.7% paraformaldehyde, blocked in 4% BSA and incubated in anti Ki67 primary antibody (Novus Biologicals, 1:50 in 1% BSA in PBS), followed by an incubation in Alexa488 anti-rabbit (Invitrogen, Molecular Probes, 1:300). Nuclei were counterstained with Toto3 (Invitrogen Molecular Probes, 1:5000). Slides were mounted in Vectashield mounting media (Vector labs), sealed and analyzed under Zeiss LSM 510 Meta confocal microscope. Images were acquired using the LSM software and the percentage of cells in G0 phase was calculated from at least 200 cells as the number of Ki67 negative cells versus all cells stained by Toto3. 2.4. FTIRM data collection Upon trypsinization, B16 mouse melanoma cells or U2OS human osteosarcoma cells were resuspended in NaCl 0.9% physiological solution, to a final concentration of approximately 1 × 106 cells/mL. Microfluidic devices were obtained by UV-lithography on 0.5 mm thick CaF2 optical windows. For all of them, the optical path was set to 8 m. Details on the fluidic chip geometries and on the fabrication protocols used are described elsewhere [17,23]. Note that both B16 and U2OS cells have been measured immediately after trypsinization for ∼2 h, without allowing them to adhere onto the substrate. Under these conditions, both cell types are spheroids, the average diameter of which is in the range of 10–15 m, slightly bigger that the optical path constrained by the fluidic cell. Therefore, contributions to spectral intensity modulation from possible cellular thickness variations during cell cycle progression are minimal. IR microspectra of individual cells were acquired at SISSI beamline [24] at Elettra – Sincrotrone Trieste, which is equipped with a Vis-IR microscope Hyperion 3000 coupled to a Vertex70 interferometer (Bruker Optics GmbH, Ettlingen, Germany). Microscope knife-edge apertures were set in order to fit the size of each cell (from 15 × 15 to 40 × 40 m). A Mercury-CadmiumTelluride detector with a 100 m sensitive element was used. 256 scans were averaged for each measurement in the wavenumber region 6000–800 cm−1 in transmission mode using a 15× condenser/objective at a spectral resolution of 4 cm−1 . BlackmanHarris 3-term apodization function and filling factor of 2 were chosen. For each cell measured, a buffer point close to the cell was also collected. Both cell and buffer spectra were rationed against air background. Each measurement run lasted no more than two hours in order to guarantee cell synchronization and avoid spectral artifacts derived from buffer waste. For the purpose of the paper, it has to be highlighted that the calibration dataset has been measured several months before the validation experiments, using silicon-like BaF2 devices (1 mm + 1 mm window thickness, 8 m optical path) [17], while for the validation datasets devices made on raw CaF2 (0.5 + 0.5 mm windows thickness, 8 m optical path) were used. This was done in order to verify the possible effects on the intensity of the phosphate bands due to the lower transparency of CaF2 substrates below 1100 cm−1 with respect to BaF2 . The consistency among calibration and validation data allows concluding that the different transmittance of these substrates has negligible effects on the reliability
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and reproducibility of the results, stated the comparable refractive index among the two [25].
2.5. FTIRM data analysis Water medium spectral contribution was subtracted from each raw cell microspectrum by using the in-house optimized Matlab routine described by Vaccari et al. [26]. Before spectral subtraction, the atmospheric compensation and offset correction routine of OPUS 6.5 (Bruker Optics GmbH, Ettlingen, Germany) were applied. Spectra from the B16 calibration dataset (SynG0, SynS and Asyn) were independently analyzed using a multivariate approach in R environment, HyperSpec [27,28] and stats packages. Briefly, Hierarchical Cluster Analysis (HCA) was performed on the absorbance spectra, based on Euclidean distances and Ward’s classification algorithm, in the 1300–1000 cm−1 spectral region. The number of clusters was chosen by cutting off the dendrograms according to the level of heterogeneity and the number of cell cycle phases identified by FC. The assignment of each cluster to a specific phase of the cell cycle was done cross-checking the consistency of the percentages among FC and FTIRM data. The reader is referred to [16] for more details. The same procedure was applied for the samples of U2OS human osteosarcoma cells. Calibration intervals for both cell lines have been then calculated as the average ± standard deviation of the area integral of the PhI band (1274–1182 cm−1 ) of all the files belonging to the same stage of the cell cycle, as assessed by FTIRM. For the B16 validation datasets, the area integral of the phosphate asymmetric stretching bands (PhI, 1274–1182 cm−1 ) was computed using OPUS 6.5 software. FTIRM histograms of the distribution of the PhI band intensity were calculated with Origin 8.1 with a binning of 0.02 a.u. and the cell cycle phase distribution assessed on the basis of the calibration intervals. One-way analysis of variance, ANOVA, has been performed with Origin 8.1. For the evaluation of the differences among the spectral profiles of mitotic cells, second derivatives of the subtracted spectra were computed by applying Savitzky–Golay algorithm (13 smoothing points) and vector normalized (OPUS 6.5 software). HCA on second derivatives has been performed in the 1200–950 cm−1 spectral region (Euclidean distances and Ward’s classification algorithm) using HyperSpec [27,28] and stats packages in R environment.
3. Results and discussion
Therefore we tentatively linked the difference in the total NA content to the diverse RNA content between G0 and G1 phases [3,29]. In order to confirm this hypothesis, B16 mouse melanoma cells from SynG0 and Asyn of calibration datasets have been immunostained for Ki67 protein, for evaluating the percentage of G0-quiescent cells. To this aim, at least 200 cells were counted for each set, an amount comparable with the cells sampled by FTIRM. Ki67 protein is a cellular marker for proliferation, expressed during all progressive phases of the cell cycle, but absent for cells in G0. Immunostaining combined with FC assessment of PI-stained DNA revealed that almost 90% of the entire population of SynG0 was induced to quiescence, while only a small fraction of the Asyn one (∼30%) was resting. An example of the Ki67 immunostaining is shown in Fig. 2, where the results for Asyn and a SynG0 are compared. Since SynG0 calibration dataset is almost entirely constituted by cells in G0 phase, in this analysis it is still considered as a unique cluster. On the contrary, the dendrogram for Asyn calibration dataset, shown in Fig. 1b, was further cut, and we could distinguish two sub-clusters of Asyn G0/G1: the larger one (∼60% of the G0/G1 population) was tentatively assigned to G1 phase (Asyn G1), the smaller (∼40% of the G0/G1 populations) to G0 (Asyn G0). The consistency of the assignment was cross-checked by calculating the average intensity of the area integral of the PhI band for the spectra belonging to the two clusters, that was lower for Asyn G0 than for Asyn G1, confirming our hypothesis. In the end, all spectra assigned to the same phase of the cell cycle (as established by FTIRM of live B16 cells: G0, G1, S and G2/M), have been merged, and the average value of the PhI band intensity has been calculated. The results are summarized in Table 1 and show that differences in NA content among the groups were statistically significant (one-way analysis of variance, ANOVA, p = 0.05). Ranges of variability of the PhI band area integral can be identified for G0, G1, S and G2/M phases, defined as the mean value for each stage minus (Min value) and plus (Max value) standard deviations, and they are potentially useful as calibration intervals to estimate the belonging of an individual B16 cell to a cell cycle stage, exclusively on the basis of its NA content. In order to validate this hypothesis, three different samples have been measured by FTIRM in CaF2 -based devices 8-m thick: B16 cells asynchronous (Asyn validation dataset from now on), B16 cells synchronized in G0 phase by growing to confluence (SynG0 validation dataset from now on) and mitotic cells (M validation dataset hereon). The distribution of the cell cycle stages for each validation dataset was then established exploiting Calibration Intervals and the classification verified a posteriori by PI-FC and, for SynG0 validation dataset, also by immunohistochemistry using Ki67 staining.
3.1. Methodology validation The first objective of this paper is to demonstrate that the cell cycle phase of an individual live cell can be assessed by FTIRM on the base of its NA content, provided that the ranges of variability of the PhI band related to each stage of the cellular progression are defined. To this aim, we first refined the data analysis of the set of data reported in [16], named calibration dataset hereafter. In that paper, we pointed out that the mean NA content for SynG0 G0/G1, established on the basis of the area integral of the PhI band (1274–1182 cm−1 ), was almost two-thirds of the one established for the same stage of asynchronous population (Asyn G0/G1), 0.210 ± 0.035 and 0.309 ± 0.050 respectively, possibly due to a different ratio between G1 and G0 cells in the two samples. Indeed, a higher percentage of quiescent cells upon growth to confluence is expected [3,29]. The RNA content of G0 cells is lower than in any other progressive stage of the cell cycle and it is almost half of that in G1 [30]. The level of ribosomal RNA is particularly low because the protein translation is less active [31].
3.1.1. Asyn validation dataset FTIRM of live asynchronous B16 cells, Asyn validation dataset, has been performed as described in Section 2. According to the area integral of the PhI band, it was established that 29% of the measured cells reside in the G0 calibration interval, 31% in the G1, 28% and 12% in the S and G2/M, respectively. Fig. 3c shows the recurrence of PhI integral area values for all the cells of the Asyn validation dataset. PI-FC gave the following output: G0/G1 53.4%; S 35.1%; G2/M 11.5% (see Fig. 3a). FTIRM and FC results are in good agreement. 3.1.2. SynG0 and M validation datasets FTIRM analysis of SynG0 B16 cells of the validation dataset allowed to determine that 51% of the cell were proliferating (35% G1, 15% S, 1% G2/M), while 49% lasted in the quiescent G0 phase (see Fig. 3c). From FC, it was possible to establish that a small percentage of cells were in S (∼9%) and G2/M phase (∼8%) upon synchronization by growth to confluence and all other (∼83%) were in G0/G1 (see Fig. 3b). Combining immunostaining for Ki67, we determined
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Fig. 2. Identification of B16 non-proliferating cells by immunostaining for Ki67. (a, d) Ki67 stained in green for SynG0 and Asyn cells; (b, e) nuclei stained with Toto3 in purple for SynG0 and Asyn cells; (c, f) merge of Ki67 and Toto3 staining for SynG0 and Asyn cells (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
that ∼77% of the cells were in G0. FTIRM data, plotted in Fig. 3d, reveal enrichment in the G0 phase with respect to the progressive phases of the cell cycle when compared to Asyn validation dataset (see Fig. 3c), while it was not possible to precisely reproduce the proportion between G1 and G0 phases obtained with Ki67 staining. Indeed, the transition from G0 to G1 is smooth, without a clear border among the two stages in terms of total NA content, but it is characterized by an intermediate population of post-mitotic cells that are increasing their RNA concentration above the restriction point for DNA replication, the early G1 cells. Those cells should have a total NA content in between G0 and late G1 cells. For verifying this point, a M validation dataset was built by enriching a B16 population in the mitotic phase using nocodazole, as described in Section 2. The enrichment of the M-phase was only ∼20% (see Fig. 4a), but it was enough for allowing an easier visualization and faster selection of mitotic cells based on morphology. As a matter of fact, for the purpose of the experiment, only cells characterized by membrane invagination, possibly in the stage of mitosis before cytokinesis,
were selected (see examples in Fig. 5, inset) and measured. For this sample, a histogram of the distribution of the PhI band was also generated and it is shown Fig. 4b. Almost all selected cells fall in the G2/M calibration interval, and all of them are represented by the Gaussian distribution of the PhI band for G2/M calibration dataset. On the background of Fig. 4b, the recurrence of the PhI of the G2/M calibration dataset is plotted in light gray, in order to better appreciate this behavior. The average value of the PhI band area integral for these cells was of 0.595 ± 0.061 a.u.. It is known that, when cells divide, daughter cells become two early G1 cells [3], and it is therefore not surprising that half of the area integral value of the PhI band for the M validation dataset is 0.297 ± 0.061 a.u., that is located in between G0 and late G1. Summarizing, the validation experiments here reported demonstrate that the cell-cycle phase distribution of B16 cells can be defined using FTIRM, only on the basis of the PhI area integral once established a calibration dataset, with a degree of accuracy comparable to FC. Moreover, the enrichment of a cell population in G0 can
Table 1 In column three (average value ± sd (a.u.)), the average values of the PhI band area integral obtained merging all the spectra of each cluster of the calibration dataset (second column, clusters) assigned to the same phase of the cell cycle (first column, phase) are reported. Differences in the NA content among the phases were statistically significant for p = 0.05 (one-way analysis of variance, ANOVA). Calibration Intervals, calculated as the average value of each phase plus/minus standard deviation are reported in the fourth column. Calibration intervals Phase
Clusters
G0
Asyn G0 Syn G0 Asyn G1 Asyn S SynS S Asyn G2/M SynS G2/M
G1 S G2/M
Average value ± SD (a.u.)
Calibration interval (a.u.)
0.233 ± 0.066
0.167–0.300
0.354 ± 0.054 0.479 ± 0.074
0.300–0.408 0.408–0.549
0.676 ± 0.127
0.549–0.803
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Fig. 3. Asyn and SynG0 Validation dataset for B16 cells – recurrence of PhI band. (a) DNA histogram and cell cycle distribution retrieved from PI-FC of the Asyn validation dataset. The underlying distribution is recovered by fitting the G0/G1 (gray) and G2/M (blue) peaks as Gaussian curves and the S phase (red) as a Gaussian-broadened curve; (b) DNA histogram and cell cycle distribution retrieved from PI-FC of the SynG0 validation dataset. The underlying distribution is recovered as in (a); (c) Recurrence of PhI integral area values for Asyn validation dataset and cell-cycle distribution evaluated according to calibration intervals. Histogram bars (binning 0.05 a.u.) in G0 calibration interval are black, in G1 green, in S red and in G2/M blue; (d) Recurrence of PhI integral area values for SynG0 validation dataset and cell-cycle distribution evaluated according to calibration intervals. The same color code of (d) is used. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
also be established, while the assessment of G0 versus G1 percentages with a degree of accuracy comparable to PI-FC assisted by Ki67 staining is hindered by the smooth increase of RNA levels between quiescent and progressive stages. 3.2. Effect of nocodazole The main advantage in using FTIRM as an alternative technique for the determination of the cell cycle phase distribution is the possibility to perform an in situ sorting of live cells without the need of labeling, staining or fixation of the sample. FTIRM indeed does
not affect the viability of the cells and moreover provides additional information on the biochemical content of living cells apart from nucleic acids. We have recently demonstrated that fixation protocols may alter the biochemical content and spectral profile of a cell, in particular in the nucleic acids region [26]. Moreover, there are several papers that report and/or warn on the possible adverse effects of synchronization protocols on cellular biochemistry [32,33]. Therefore, the availability of a label-free technique for the in vitro determination of the cellular phase could benefit modern cellular biology. As an example, we investigated the effects of synchronization protocols on cellular biochemistry by comparing
Fig. 4. M Validation dataset for B16 cells (a) DNA histogram and cell cycle distribution retrieved from PI-FC of the M validation dataset. The underlying distribution is recovered by fitting the G0/G1 (gray) and G2/M (blue) peaks as Gaussian curves and the S phase (red) as a Gaussian-broadened curve; (b) Recurrence of PhI integral area values for mitotic cells of M validation dataset. Histogram bars (binning 0.05 a.u.) in S calibration interval are in red and in G2/M in blue. On the background, the recurrence of the PhI of the G2/M calibration dataset is plotted in light gray. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 5. Effects of the different synchronization protocols in M phase (a) HCA performed on vector normalized second derivatives of mitotic cells coming from three different preparations (i) synchronization by nocodazole, (ii) synchronization by G1 block (using thymidine and aphidicolin) and release to M-phase, and (iii) mitotic cells from an asynchronous population. Two main clusters are obtained, one made by nocodazole treated cells and the other by mitotic cells from normally progressive populations. The inset shows the type of cells chosen for the measurements by morphologycriteria; (b) cluster centroids related to nocodazole treated cells (red) and the other two preparations (blue). Line thickness is proportional to the standard deviation of each centroid (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
spectra of mitotic live cells following three different approaches: (i) synchronization by nocodazole, (ii) synchronization by G1 block (using thymidine and aphidicolin) and release to M-phase, and (iii) mitotic cells from an asynchronous population. Cell spectra have been selected according to the shape criteria defined in Section 3.2. Fig. 5a reports the results of HCA analysis performed on the second derivatives of the subtracted spectra for the three M groups in the 1200–950 cm−1 spectral region. Despite the low spectral heterogeneity levels, two main clusters are detected: the first one constituted of mitotic cells treated with nocodazole, the second of mitotic cells from G1block and asynchronous populations. Cluster centroids are shown in Fig. 5b. Some specific differences for the enrichment with nocodazole are detected. In particular, a displacement from 1049 to 1053 cm−1 and from 1014 to 1025 cm−1 of the C O stretching coupled with C O bending of C OH moieties of cellular carbohydrates (see Fig. 5b) [34–36]. Moreover, the variation of the spectral shape in the range 1165–1145 cm−1 could be possibly assigned to C O stretching of C OH groups [37] and therefore related to variations in cellular carbohydrates. All these spectral variations could be tentatively correlated to the mode of action of nocodazole. Nocodazole blocks the progression of mitosis by inhibiting the formation of microtubules [38] and it has also been reported to disturb the transport of newly synthesized proteins [39]. Moreover, it has been found that arresting cells in G2/M using anti-microtubule agents (such as nocodazole) causes a
noticeable increase in the concentration of N-acetylglucosamine (GlcNAc) residues on numerous glycoproteins [40]. This altered glycosilation was found to be independent from microtubule disassembly and not directly related to G2/M phase of the cell cycle [41]. This is in agreement with the observation that normally progressing cells (Asyn G2/M and M release from G1) cluster together, while changes in glycosilation may be responsible for the shifts observed in the spectral contributions of cellular sugars in the 1200–950 cm−1 spectral region, as also reported by other authors [42]. 3.3. Assessment of the cell-cycle distribution of U2OS cell line by FTIRM The possibility to discriminate among different stages of the cell cycle by FTIRM on the basis of the NA content might have a general applicability for mammalian cells. Indeed, despite the specific genomic profile of each cell line, the relative variations of cellular DNA and RNA follow comparable trends during cellular progression. In order to demonstrate that the proposed methodology could be extended to other cell lines, it was applied to analyze the phase cycle distribution of U2OS human bone osteosarcoma cells. Specifically, we built a test dataset for U2OS cells following a simplified approach with respect to the one implemented in [16], and summarized in the introduction. Only asynchronous
Fig. 6. Calibration dataset for U2OS. (a) DNA histogram retrieved from PI-FC analysis of a representative Asyn sample of the U2OS test dataset. The underlying distribution is recovered by fitting the G0/G1 (gray) and G2/M (blue) peaks as gaussian curves and the S phase (red) as a gaussian-broadened curve; (b) Hierarchical Cluster Analysis (HCA) of absorbance spectra based on Euclidean distances and Wards’ classification algorithm in the 1300–1000 cm−1 spectral region for the U2OS Asyn test dataset. In the inset, the percentages of the cells belonging to each cluster and the cluster assignment are reported; (c) Plot of the spectral centroids of each cluster ± standard deviation (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Table 2 Cell cycle phase distribution obtained for U2OS human osteosarcoma cells using FC with PI staining averaged on a triplicate experiment is reported in column “FC results”; cell cycle phase distribution obtained by Hierarchical Cluster Analysis (HCA) of absorbance spectra based on Euclidean distances and Wards’ classification algorithm in the 1300–1000 cm−1 spectral region for the same U2OS cell population is reported in column “HCA results”. Area integral of the PhI band ± standard deviation is also reported for each cluster centroid in column “Average value ± sd (a.u.)”. Differences in the NA content among the phases were statistically significant for p = 0.05 (one-way analysis of variance, ANOVA). Calibration Intervals, calculated as the average value of each phase plus/minus standard deviation are also reported in the last column (Calibration Interval (a.u.)). Phase
FC results (%)
HCA results (%)
Average value ± sd (a.u.)
Calibration interval (a.u.)
G0/G1 S G2/M
41.5 ± 12.0 19.5 ± 0.7 39 ± 12.7
45.90 23.50 30.60
0.181 ± 0.044 0.264 ± 0.043 0.324 ± 0.064
0.137–0.226 0.220–0.307 0.256–0.389
populations of U2OS cells have been taken into account, in order to make the protocol faster. Parallel FTIRM and PI-FC experiments on an asynchronous population of U2OS cells has been done, and repeated in triplicate. PI-FC analysis allowed to establish the following distribution: G0/G1 41.5 ± 12.0%, S 19.5 ± 0.7%, G2/M 39.0 ± 12.7% (standard deviations are related to the triplicate). Fig. 6a shows the FC histogram for one of the Asyn U2OS samples. HCA on absorbance microspectra of the entire test dataset in the 1300–1000 cm−1 spectral region provided the dendrogram shown in Fig. 6b. The dendrogram was cut into three clusters, according to FC results. Clusters were then assigned to the G0/G1, S and G2/M phases conforming to the relative percentages. 45.9% of the cell population was classified in the G0/G1 phase by FTIRM, 23.50% in S and 30.6% in G2/M. The correctness of the assignment was checked by retrieving the average NA content per each cluster, defined as the average of the area integral of the PhI band for all the microspectra belonging to it (see Fig. 6c). In agreement with the results obtained for B16 cells, the intensity of this band almost doubled from G0/G1 to G2/M (see Table 2), confirming the predictive power of the proposed methodology also for U2OS cells and the comparable accuracy of FTIRM and PI-FC.
4. Conclusions In this work, we showed that, once built a calibration dataset for assessing the cell cycle stages of live B16 melanoma cells and defined calibration intervals based on the average intensity of the PhI band, these can be exploited in subsequent experiments for determining the status of any individual live B16 cell, with a degree of accuracy comparable to FC determination with PI staining of G0/G1, S and G2/M phases. In addition, it was possible to verify that FTIRM may discriminate to some extent between G0 and late G1 cells. These results might have a general value for mammalian cell line, as proved for U2OS human bone osteosarcoma cells. FTIRM can be performed on live cells, which can be therefore sorted within the fluidic device according to their progression state. Those cells can be then tested for their individual response to chemical–thermal–mechanical stresses, being able to assert the response specificity linked to the cell-cycle stage, following a totally label-free experimental approach. The FTIRM-based method here proposed does not require the enrichment of the cell population to a specific cell-cycle stage through synchronization protocols. Indeed, synchronization protocols may induce biochemical changes, and consequent spectral variations, not directly related to the specific cell-cycle stage, but to the procedures followed for population enrichment. Actually, specific changes induced by nocodazole in B16s were found, when comparing to mitotic cells for normally progressive populations. Therefore, the possibility to simply identify the cell cycle phase of an individual cell on the basis of its NA content by FTIRM is of paramount importance and offers new perspectives in many fields of science where this knowledge is fundamental.
Acknowledgements This study was partially financed from the Italy–Slovenia CrossBorder Cooperation Programme, 2007–2013, project Glioma. The authors thank Dr. Alessandro Marcello for support on flow cytometric measurements.
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