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Abstract
In this work, we report that the effect of bioactive constituent on living glioma cells can be evaluated using terahertz time-domain attenuated total reflection (THz TD-ATR) spectroscopy in a label-free, non-invasive, and fast manner. The measured THz absorption coefficient of human glioma cells (U87) in cell culture media increases with ginsenoside Rg3 (G-Rg3) concentration in the range from 0 to 50 µM, which can be interpreted as that G-Rg3 deteriorated the cellular state. This is supported either by the cell growth inhibition rate measured using a conventional cell viability test kit or by the cellular morphological changes observed with fluorescence microscopy. These results verify the effectiveness of using the THz TD-ATR spectroscopy to detect the action of G-Rg3 on glioma cells in vitro. The demonstrated technique thus opens a new route to assessing the efficacy of bioactive constituents on cells or helping screen cell-targeted drugs.
© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement
Corrections
5 April 2022: A minor correction was made to Reference 33.
1. Introduction
As the global population ages, the treatment of cancer is critical to the quality of people’s life and the social economy [1]. In recent years, bioactive chemicals from plants and herbs have attracted tremendous attention from researchers who have been endeavoring to gain new inspiration and develop new therapeutic phytochemical drugs [2]. For example, the role of docetaxel, curcumin and ginsenoside has been extensively explored, which greatly enhanced our understanding of the actions and underlying mechanisms of natural medical drugs on cancer treatment [3–5].
Cell analysis plays a pivot role in biomedical and pharmacological research [6]. Normally, monitoring the effect of drugs on cells is a crucial step in the process of discovering/screening new drugs or probing the underlying biological mechanism of drug functions [7–9]. Conventionally, the effect of drugs on cells was assessed using cell labeling/staining techniques such as CCK-8 and MTT cell proliferation assays [3,10]. Despite the wide application of these techniques, it has been gradually recognized that the labeling/staining method could potentially disturb the physiological status of living cells due to introducing of external chemicals into the cells [11,12]. Besides, the labeling/staining approach costs extra money and is time-consuming. As a result, a label-free (including stain-free) technique that can be used to assess cell state in a quick, cheap, and convenient manner is highly demanded.
Recently, terahertz time-domain attenuated total reflection (THz TD-ATR) spectroscopy has been widely accepted as a label-free technique for cell study [13–15]. Terahertz (THz) wave refers to the electromagnetic radiation with a frequency ranging from 0.1 to 10 THz (1 THz = 1012 Hz) [16–19]. As THz wave has a high sensitivity to biomolecules and a relatively low photon energy, THz time-domain spectroscopy (THz-TDS) has been regarded a powerful label-free and nondestructive technique for biological samples [20–26]. The combination of THz-TDS with the attenuated total reflection (ATR) technique, namely, THz TD-ATR spectroscopy, makes it feasible to measure the interaction between the sample and the evanescent THz wave, by which the detection sensitivity is remarkably enhanced, and the adverse influence of the exceptionally high THz absorption of polar solvent (e.g., water) is minimized [27–29]. As a result, the THz TD-ATR spectroscopy has a performance superior to the conventional transmission or reflection mode THz-TDS in measurement of biological samples in an aqueous environment [14]. By taking the advantages of THz TD-ATR spectroscopy, researchers have carried out quite a few excellent studies in monitoring the physical and biological properties of cells. For example, the dielectric characteristics and permeabilization of cells, and cell death and types were successfully investigated [13,14,27–29]. Unfortunately, although rich information was accumulated in terms of using THz wave to measure cell properties, little work has been carried out to interrogate the actions of bioactive chemicals on living cells using THz TD-ATR spectroscopy, limiting potential applications of THz TD-ATR spectroscopy in the pharmacological field.
Ginseng is a famous herbal medicine with a variety of therapeutic and pharmacological effects on human health conditions [30–34]. Ginsenoside Rg3 (G-Rg3, Fig. 1) is one of the important bioactive phytochemicals of ginseng, and is believed having anti-cancer functions [30,35,36]. However, hitherto the knowledge on the effect of G-Rg3 on cancer cells is still very limited, and the action of G-Rg3 against living cells was mainly examined with conventional labeling/staining techniques. Therefore, it is imperative to establish a THz TD-ATR spectroscopy-based technique to investigate the influence of G-Rg3 on living cells.
Fig. 1. Chemical structure of G-Rg3. The ball-and-stick model and the skeletal formula of G-Rg3 are shown on the left and right, respectively.
As the most common primary brain cancer, gliomas are terrible tumors that occur most prevalently in adults [37]. In the present work, we studied the effect of G-Rg3 on human glioma cells (U87) in cell culture media using the THz TD-ATR spectroscopy. By analyzing the measured THz spectroscopy, it was found that the measured THz absorption coefficient of U87 cells increases with G-Rg3 dose from the range of 0 to 50 µM. This phenomenon can be interpreted as that the state of U87 cells was deteriorated by G-Rg3 in a dose-dependent manner, based on both the cell growth inhibition rate measured using the CCK-8 assay and the cellular morphological alterations observed by fluorescence microscopy. Taken together, we demonstrated that the THz TD-ATR spectroscopy technique can be used to reliably probe the actions of natural bioactive chemical on living cells in a near-physiological environment in a label-free, non-invasive, and convenient manner.
2. Experimental methods
2.1 Sample preparation
G-Rg3 powder, with purity better than 98% in mass fraction, was ordered from Beijing Solarbio Science & Technology Co., Ltd., Beijing, China. The stock solution of G-Rg3 with a concentration 20 mg/mL was prepared by dissolving G-Rg3 in dimethyl sulfoxide (DMSO, analytical grade, Sigma-Aldrich China, Shanghai, China) and stored in a fridge at 4°C. The culture media is composed of a mixture (v/v) of 89% Dulbecco’s Modified Eagle’s Medium (DMEM), 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin. DMEM, FBS, penicillin-streptomycin, trypsin-EDTA and phosphate buffer solution (PBS, pH 7.4) were purchased from Gibco, Life Technology, Shanghai, China. A commercial cell counting kit (CCK-8) used for cell viability test was obtained from Dojindo Molecular Technologies, Inc., Kumamoto Prefecture, Kyushu, Japan. Paraformaldehyde was ordered from Wuhan Servicebio Technology Co., Ltd., Wuhan, China. Triton X-100 and bovine serum albumin (BSA) were provided by Sigma-Aldrich, St. Louis, Missouri, USA; Phalloidin and DAPI were obtained from Thermo Fisher Scientific, Waltham, Massachusetts, USA. Paraformaldehyde, Triton X-100 and BSA were diluted in PBS to achieve desired concentrations, respectively. Cell adhesion promoting supplement was purchased from Shanghai BasalMedia Technologies Co., Ltd., Shanghai, China. Other disposable consumables such as flasks, petri-dishes, pipet and tubes used in the experiment were provided by Corning Inc., New York, USA.
2.2 Cell culture
Human glioma cells (U87) were purchased from CHI Scientific Inc., Shanghai, China [37]. The cells were seeded in tissue culture flasks that were kept in an incubator to grow in a 5% CO2 humidified atmosphere at 37°C. The growth state of cells was monitored by an optical microscope (IX53, Olympus Co., Tokyo, Japan). The culture media were replaced every other day until the cells reached ∼80% confluence. Then, the culture media were removed before the cells were washed with 2 mL of PBS. Afterwards, the cells were treated with 2 mL of 0.25% trypsin-EDTA for 2 min, followed by adding 4 mL of cell culture media to neutralize the trypsin-EDTA for 30 s. Next, a pipet was used to gently mix the solution containing cells for several times to detach the cells from the flask wall. Later, the cell solution was collected and centrifuged at 3,000 rpm for 1 min. Finally, the pellet was re-suspended in cell culture media for use in later experiments.
2.3 Cell growth inhibition rate assessment
The growth inhibition rate of U87 cells upon exposure to G-Rg3 was evaluated by CCK-8 assay, according to the manufacturer’s instruction [3]. The cells (1×105 cell/mL) were plated in 96-well microplates (100 µL/well) to grow for 24 h in the incubator. Before the cell treatment, G-Rg3 solutions of different concentrations (0, 12.5, 25.0, 50.0, and 100.0 µM) were prepared by diluting the G-Rg3 stock solution in the cell culture media, and the DMSO was adjusted to 0.1% of the solution (m/m) [4]. Among the G-Rg3 solutions prepared above, the solution containing no G-Rg3 (0 µM) was used as the control solution, which has been proven having no observable effect on the cell state [4,9]. Afterwards, the culture media in each well were replaced with the same volume of G-Rg3 solution of different concentrations (0, 12.5, 25.0, 50.0, and 100.0 µM), and the cells were incubated for 24 h. Later, the samples were washed once with the cell culture media (100 µL/well) to remove the residual G-Rg3, followed by adding 10 µL of CCK-8 solution and 100 µL of cell culture media to each well to allow the cells to grow for 3 h in the incubator. Finally, the microplates were measured in a microplate reader (Infinite M200pro, Tecan Group Ltd., Männedorf, Switzerland), and the optical absorbance value (OD) at 450 nm was recorded. In the CCK-8 assay, the cells treated with the control solution were used as the control group while the cells treated with G-Rg3 solutions with concentrations of 12.5, 25.0, 50.0, and 100.0 µM were used as the experimental group. The cell growth inhibition rate was presented as the percentage ratio between the experimental group and the control group, according to Eq. (1) [3]:
The experiments were performed in four replicates and repeated at least three times.
2.4 Fluorescence microscopy
U87 cells seeded in petri-dishes were incubated for 24 h before exposed to G-Rg3 solution with various concentrations (0, 25.0, and 50.0 µM) for 24 h in the incubator, respectively, then the solution was removed and the cells were washed with PBS for two times. Afterwards, the cells were fixed with 4% formaldehyde for 10 min at room temperature, and washed two times with PBS before permeabilized with 0.1% Triton X-100 for 20 min at room temperature. Following wash the samples two times with PBS, 1 mL 1% BSA was added into the petri-dish to block the Triton X-100 for 3h at ∼37°C. After washing two times with PBS, the cells were incubated in the solution containing 10 µL Alexa Fluor 488 phalloidin (ThermoFisher Scientific, Shanghai, China) and 200 µL PBS for 40 min to stain the actin filaments in dark at room temperature [38]. Later, the cells were washed two times with PBS before incubated in the solution containing 1 µL DAPI (ThermoFisher Scientific, Shanghai, China) and 1800 µL PBS for 5 min to stain the nuclear in dark at room temperature [38]. Subsequently, the cells were washed three times with PBS and submerged in 2 mL PBS. Finally, the stained cell samples were observed using a confocal microscope (FV1200, Olympus Co., Tokyo, Japan).
2.5 THz TD-ATR spectroscopy system
The THz TD-ATR spectroscopy system (Fig. 2) was developed by incorporating an ATR apparatus (BATOP GmbH, Jena, Germany) into the optical path of a commercial THz-TDS system (Tera K15, Menlo Systems GmbH, Münich, Germany) having a resolution of 1.2 GHz and a frequency range of 0.1 to 4.5 THz [39]. The ATR apparatus comprises a silicon prism and a liquid cell situated on top of the prism to contain liquid samples. A p-polarized pulsed THz beam is horizontally incident on the left slanted side of the prism, and then refracted in the prism. The refracted beam is completely reflected at the upper surface of the prism, a phenomenon called ‘attenuated total internal reflection’. Concomitantly, an evanescent field is produced at the upper surface, which can interact with the matter located on the prism base with a high sensitivity. Finally, the emergent THz beam transmitted through the right side of the prism is detected and analyzed, by which useful information of measured samples can be disclosed.
Fig. 2. Schematic illustration of the ATR apparatus. An ATR apparatus was incorporated into the optical path of a THz-TDS system to build up the THz TD-ATR spectroscopy system.
2.6 THz measurement of cells
To investigate the influence of G-Rg3 on the cells using the THz TD-ATR spectroscopy, the cells were grown in the cell culture flasks for 24 h, and then treated with the G-Rg3 solution with different concentrations (0, 12.5, 25.0, 50.0, and 100.0 µM) for 24 h, respectively. The cells were collected and re-suspended into DMEM containing 0.1% (v/v) cell adhesion promoting supplement. In THz experiments, 2 mL of cell solution was transferred to the liquid cell, and maintained for 2 min to allow the cells to attach to the ATR surface before the collection of THz data. It needs to note that the time of 2 min is the optimized adherent time when the number of cells attached the ATR prism surface reaches equilibrium in our experimental system. To minimize the THz absorption by the water vapor in the environment, the THz optical path and the ATR apparatus were sealed in chamber with a constant relative humidity of 2% achieved by continuously purging nitrogen during measurements. THz measurement of each sample can be completed within 30 s, and the liquid cell filled with nitrogen was firstly measured as the reference for data analysis. All the measurements were conducted at about 300 K, and three independent experiments were performed for each sample.
2.7 Theory of THz TD-ATR spectroscopy
To assess the response of cells to THz wave, the measured THz time-domain spectra were transformed into frequency-domain spectra by the fast Fourier transform algorithm, and the absorption coefficient ($\alpha (\omega )$) of the sample, which is a function of the THz frequency $\omega $, was calculated step by step from the frequency-domain THz spectra [39]. The absorption coefficient is a reliable physical parameter, and has much better performance than the refractive index in terms of characterizing biological samples in aqueous solution [40–42]. The procedures of calculating $\alpha (\omega )$ are described below.
First, the ratio of the electric amplitudes of the frequency-domain spectra of the sample (${\tilde{E}_{\textrm{sam}}}$) and reference (${\tilde{E}_{\textrm{ref}}}$) was calculated, which equals the ratio of the Fresnel’s reflection coefficients of the sample (${\tilde{r}_{\textrm{sam}}}$) and reference (${\tilde{r}_{\textrm{ref}}}$), as shown in Eq. (2):
Second, ${\tilde{r}_{\textrm{ref}}}$ and ${\tilde{r}_{\textrm{sam}}}$ can be calculated by Eq. (3), respectively:
For the reference (liquid cell filled with nitrogen) measured, $\tilde{r}$ in Eq. (3) is ${\tilde{r}_{\textrm{ref}}}$, and $\tilde{\varepsilon }$ is approximated as 1 for nitrogen. Thus, the value of ${\tilde{r}_{\textrm{ref}}}$ was directly calculated by Eq. (3). For the cell sample measured, $\tilde{r}$ in Eq. (3) is ${\tilde{r}_{\textrm{sam}}}$, and $\tilde{\varepsilon }$ is the complex permittivity of the cell sample in the vicinity of the prism surface. As the ratio of ${\tilde{E}_{\textrm{sam}}}$ to ${\tilde{E}_{\textrm{ref}}}$, and the value of ${\tilde{r}_{\textrm{ref}}}$ were known, the value of ${\tilde{r}_{\textrm{sam}}}$ was calculated by Eq. (2).
Third, ${\tilde{r}_{\textrm{sam}}}$ was substituted into Eq. (3) to obtain the complex permittivity $\tilde{\varepsilon }$ of the cell sample. The real part ($\varepsilon ^{\prime}$) and imaginary part ($\varepsilon ^{\prime\prime}$) of $\tilde{\varepsilon }$ can be written as:
From Eqs. (4) and (5), the refractive index ($n(\omega )$) and extinction coefficient ($k(\omega )$) were calculated.
Finally, the absorption coefficient of the cell sample was calculated by substituting the speed of light in vacuum c into Eq. (6):
3. Results and discussion
3.1 THz absorption coefficients of U87 cells
To assess the effects of G-Rg3 on U87 cells using the THz TD-ATR spectroscopy, THz absorption coefficients of the cells treated by G-Rg3 with different concentrations for 24 h were analyzed (Fig. 3). The absorption coefficients of the cells treated by G-Rg3 of different concentrations are difficult to be discriminated from each other for the frequency lower than 0.75 THz, but can be easily differentiated from each other for the frequency larger than 0.75 THz, except for those of the cells treated by G-Rg3 of higher concentrations (50 µM and 100 µM) that are nearly overlaid with each other. This means that for the frequency between 0.75 to 1.0 THz and for the G-Rg3 concentration from 0 to 50 µM, the THz absorption coefficients increase with both the frequency and G-Rg3 concentration (Fig. 3(b)).
Fig. 3. THz absorption coefficients of U87 cells treated by G-Rg3 of different concentrations for 24 h. The absorption coefficients corresponding to the frequency range from 0.3 to 1.1 THz and from 0.75 to 1.0 THz are shown in (a) and (b), respectively. The cells without suffering from G-Rg3 was used as control, i.e., the concentration of G-Rg3 is 0 µM. The error bar in (b) represents the standard deviation (SD).
By taking 0.84, 0.88 and 0.92 THz as examples, the properties of THz absorption coefficient as function of G-Rg3 concentration were examined. Interestingly, it was found that for an individual frequency (e.g., 0.84, 0.88 or 0.92 THz) the absorption coefficient of the cells treated by G-Rg3 increases with G-Rg3 concentration until 50 µM, and then flattens out (Fig. 4(a)). For different frequencies, the observed trend is very similar to each other, which can be further verified by the analysis of the absolute change of the absorption coefficients of cells treated by G-Rg3 at various concentrations, relative to that of the cells not subjected to G-Rg3 treatment (0 µM) for different frequencies, respectively (Fig. 4(b)). The phenomena shown in Fig. 4 can be observed for any frequency between 0.75 and 1.0 THz. The observation manifests that upon the exposure to G-Rg3 the state of U87 cells were changed, suggesting the action of G-Rg3 on U87 cells can be evaluated using the THz absorption coefficient.
Fig. 4. Analysis of THz absorption coefficients of U87 cells at certain frequencies. (a) THz absorption coefficients of cells at 0.84, 0.88 and 0.92 THz as function of G-Rg3 concentrations. The cells were treated with G-Rg3 at a series of different concentrations (0, 12.5, 25, 50 and 100 µM) for 24 h. The cells without suffering from G-Rg3 was used as control, i.e., the concentration of G-Rg3 is 0 µM. (b) The change of THz absorption coefficients relative to the control for 0.84, 0.88 and 0.92 THz, respectively. The error bar stands for the SD.
It needs to note that the single-interface model was used in the analysis of the THz absorption coefficient in the present work, i.e., the cell layer and the liquid medium (DMEM) were not considered separately in the data analysis [14,29]. As the penetration depth (∼ 30 µm@1 THz) of the evanescent wave is relatively larger than the height of cells (∼ 10 µm) [13,29], the calculated THz absorption coefficients (Fig. 3, and Fig. 4(a)) not only contain the contribution from the cells, but also likely contain the contribution from the liquid medium [14]. However, the change of the THz absorption coefficients relative to the control cells (Fig. 4(b)) can reliably represent the property change of the cells upon the treatment by G-Rg3, which contains comprehensive information on the cellular changes such as chemical changes, morphological variations, and structural alterations.
3.2 CCK-8 assay on U87 cells
In order to evaluate the cell state for the cells suffered from the treatment by G-Rg3, a conventional biological technique, CCK assay, was performed, and the cell growth inhibition rate was calculated. The data show that the cell inhibition rate increased from 0 to 52% when the G-Rg3 concentration increased from 0 to 100 µM (Fig. 5), indicating that the cell state has been changed indeed by G-Rg3. This result is highly consistent with the literature in which the action of G-Rg3 against U87 cells was measured by the MTT assay that can be regarded as the twin of the CCK-8 assay in terms of measuring the cell growth inhibition rate [9].
3.3 Effectiveness of THz label-free assessment of effects of G-Rg3 on U87 cells
From the results measured by the THz TD-ATR spectroscopy (Fig. 4) and CCK-8 assay (Fig. 5), it can be found that either the THz absorption coefficient or the cell growth inhibition rate increases monotonically with increasing G-Rg3 concentration in the range between 0 and 50 µM, indicating the effectiveness of using the THz absorption coefficient to characterize the state of the cells treated by G-Rg3 with a dose below 50 µM. This is further validated by the cellular morphological alterations observed by fluorescence microscopy (Fig. 6). Compared to the control (Fig. 6(a)), the morphology of cells was altered more and more evidently when the concentration of G-Rg3 increased from 25 µM (Fig. 6(b)) to 50 µM (Fig. 6(c)), as indicating by more cells changed from a spindle-like shape to a rounded-shape. This phenomenon is highly consistent with what observed by SIN et al. for G-Rg3 induced cell morphological change using an optical microscope [9]. In their work, they found that U87 cells gradually became round from a spindle-like shape with the increase of G-Rg3 concentration. According to their work, it is very likely that the cell growth inhibition at 12.5 µM and 25 µM was mainly caused by the mechanism of cellular senescence induced by the G-Rg3 treatment while the cell growth inhibition at 50 µM and 100 µM was mainly due to the mechanism of cellular apoptosis resulted from the G-Rg3 treatment.
Fig. 5. Plot of the growth inhibition rate of U87 cells upon the treatment with G-Rg3 of different concentrations for 24 h. The cells without suffering from G-Rg3 was used as control, i.e., the concentration of G-Rg3 is 0 µM. The error bar denotes the SD.
Fig. 6. Fluorescent images of U87 cells. (a-c) are cells treated by 0 µM (control), 25 µM and 50 µM G-Rg3 for 24 h, respectively. Actin filaments stained by phalloidin to green, and nuclear stained by DAPI to blue. Scale bar, 20 µm.
However, there are also some dissimilarities between the plots obtained from THz TD-ATR spectroscopy (Fig. 4) and CCK-8 assay (Fig. 5). On one hand, the plots in Fig. 4 present a convex shape while that in Fig. 5 shows a concave shape. This can mainly be attributed to that the two methods are based on different fundamental principles. The THz TD-ATR spectroscopy is a physical method of measuring the dielectric response of cells on the ATR base to THz waves while CCK-8 is a chemical technique based on characterizing the amount of formazan produced in the test cells. Therefore, the trends of the results obtained by these two methods are not necessarily to be linearly correlated, as far as they are not against each other in general.
On the other hand, unlike the cell inhibition rate measured by the CCK-8 assay that increases in the whole G-Rg3 dose range from 0 to 100 µM (Fig. 5), the THz absorption coefficient extracted from the THz TD-ATR spectroscopy reaches a plateau when G-Rg3 dose is higher than 50 µM (Fig. 4), i.e., the THz absorption coefficient for the cells treated by 100 µM is nearly the same as that of the cells treated by 50 µM. This discrepancy may originate from the different sample preparation procedures used in THz TD-ATR spectroscopy and CCK-8 measurements. For the former the cells were treated by G-Rg3 first, then the cell solution was loaded onto the ATR prism to allow cells to attach to the prism surface prior to the measurement; whereas for the latter the cells were grown in the wells of microplates, treated by G-Rg3, and then measured after removing the residual G-Rg3. It is known that cells with poor viability normally are difficult to attach to a surface from a cell suspension solution. In our case, it could be that the U87 cells in too bad state upon the treatment by 100 µM G-Rg3 failed to attach to the ATR prism surface to be measured. Therefore, the cells attached to the ATR prism surface have similar properties for the samples treated by 50 µM G-Rg3 and 100 µM G-Rg3. Consequently, the sample of the cells treated by 100 µM G-Rg3 has similar THz absorption coefficient as that of the cells treated by 50 µM G-Rg3. This observation may suggest that there is a saturate threshold in terms of drug dose in THz TD-ATR spectroscopy measurement. In practice, it is not necessarily to use a very high dose in the investigation of drug-cells interactions, particularly in the screening of drugs, so the THz TD-ATR spectroscopy approach can be effectively applied in biochemical and pharmacological areas. It needs to note that in contrast to the CCK-8 assay introducing external chemicals into cells and taking several hours before measurement, the THz TD-ATR spectroscopy technique does not require introducing any chemicals into cells, and only took a few minutes to evaluate the effects of G-Rg3 on a cell sample. These label-free, non-invasive, and time-saving features make the THz TD-ATR spectroscopy technique very promising in practical applications.
Previous studies indicate that factors such as chemicals (e.g., protein and ion) inside cells as well as cellular morphology/coverage can contribute to the THz absorption coefficient of measured cells [13,14]. In our case, based on the results from CCK-8 assays and fluorescent studies, we are sure that the changes in both chemicals and morphology of the cells contributed to the observed phenomenon of the THz absorption coefficient changing with the G-Rg3 concentration from 0 to 50 µM. Nevertheless, due to the intrinsic complexity of the interaction between cells and THz wave, it is worthwhile for researchers to carry out more experiments to further investigate how the possible factors influence the THz absorption of cells.
4. Conclusion
In summary, we demonstrated a THz TD-ATR spectroscopy technique that can be used to reliably assess the influence of G-Rg3 on U87 cells in vitro in a label-free, non-invasive, and fast manner, as far as the dose of G-Rg3 is not beyond the saturate threshold dose of 50 µM. The effectiveness of this approach was confirmed by both the cell viability test (CCK-8) and the cellular morphological alterations (fluorescent images). Considering the urgent needs in label-free and convenient detection of cell states and screening anti-cancer drugs, this work provides a new promising method for the assessment of drug actions on the cell level, which is a crucial step in the development of new drugs. Undoubtedly, this technique can find a spectrum of applications in the future.
Funding
National Key Research and Development Program of China (2016YFD0400800, 2021YFA1301503); National Natural Science Foundation of China (11079019, 12074208, 62175238, U1932132); the University of Chinese Academy of Sciences Supported Program for Tackling Key Problems in Science and Technology (E029610601); Natural Science Foundation of Chongqing (cstc2019jcyj-msxmX0654); Beibei District Science and Technology Agency Supported Program for Science and Technology Talents & Independent Innovation (2021-6).
Disclosures
The authors declare that there are no conflicts of interest related to this manuscript.
Data Availability
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.
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