52 research outputs found
Quantitative phase microscopy timelapse dataset of PNT1A, DU-145 and LNCaP cells with annotated caspase 3,7-dependent and independent cell death
No full text<p>Time-lapse dataset of prostatic cell lines (DU-145, PNT1A, LNCaP) exposed to cell death-inducing compounds (staurosporine, doxorubicin) and black phosphorus. The time-lapse dataset is annotated as follows: (1) cell masks and cell numbers, (2) by cell death type and timepoint of death in the attached xlsx file. This dataset is supplementary to the article:</p>
<p>Vicar, T., Raudenska, M., Gumulec, J. <em>et al.</em> The Quantitative-Phase Dynamics of Apoptosis and Lytic Cell Death. <em>Sci Rep</em> <strong>10, </strong>1566 (2020). <a href="https://doi.org/10.1038/s41598-020-58474-w">https://doi.org/10.1038/s41598-020-58474-w</a></p>
<p>Correlative fluorescence microscopy is in a separate dataset <a href="https://doi.org/10.5281/zenodo.4531900">10.5281/zenodo.4531900</a></p>
<p>Code is available at <a href="https://github.com/tomasvicar/CellDeathDetect">https://github.com/tomasvicar/CellDeathDetect</a></p>
<p><strong>Methods</strong></p>
<p><em>Cell culture and cultured cell conditions</em><br>
LNCaP cell line was established from a lymph node metastase of the hormone-refractory patient and contains a mutation in the AR gene. This mutation creates a promiscuous AR that can bind to different types of steroids. LNCaP cells are AR-positive, PSA-positive, PTEN-negative and harbor wild-type p53 {Skjoth, 2006 #150; Mitchell, 2000 #149}. PNT1A is immortalized non-tumorigenic epithelial cell line. PNT1A cells harbour wild-type p53. However, SV40 induced T-antigen expression inhibits the activity of p53. This cell line had lost the expression of androgen receptor (AR) and prostate-specific antigen (PSA) (Raudenska, 2019). DU-145 cell line is derived from the metastatic site in the brain and contains P223L and V274F mutations in p53. This cell line is PSA and AR-negative and androgen independent (Chappell, 2012). All cell lines used in this study were purchased from HPA Culture Collections (Salisbury, UK). and were cultured in RPMI-1640 medium with 10 % FBS. The medium was supplemented with antibiotics (penicillin 100 U/ml and streptomycin 0.1 mg/ml). Cells were maintained at 37°C in a humidified (60%) incubator with 5% CO2 (Sanyo, Japan).</p>
<p><em>Correlative time-lapse quantitative phase-fluorescence imaging</em></p>
<p>QPI and fluorescence imaging were performed by using multimodal holographic microscope Q-PHASE (TESCAN, Brno, Czech Republic). To determine the amount of caspase-3/7 product accumulation, cells were loaded with 2 µM CellEventTM Caspase-3/7 Green Detection Reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol and visualized using FITC 488 nm filter. To detect the cells with a loss of plasma membrane integrity, cells were stained with 1 ug/ml propidium iodide (Sigma Aldrich Co., St. Louis, MO, USA) and visualized using TRITC 542 nm filter. Nuclear morphology and chromatin condensation were analyzed using Hoechst 33342 nuclear staining (ENZO, Lausen, Switzerland) and visualized using DAPI 461 nm filter. Cells were cultivated in Flow chambers μ-Slide I Lauer Family (Ibidi, Martinsried, Germany). To maintain standard cultivation conditions (37°C, humidified air (60%) with 5% CO2) during time-lapse experiments, cells were placed in the gas chamber H201 - for Mad City Labs Z100/Z500 piezo Z-stages (Okolab, Ottaviano NA, Italy). To image enough cells in one field of view, lens Nikon Plan 10/0.30 were chosen. For each cell line and each treatment, seven fields of view were observed with the frame rate 3 mins/frame for 24 or 48 h respectively. Holograms were captured by CCD camera (XIMEA MR4021 MC-VELETA), fluorescence images were captured using ANDOR Zyla 5.5 sCMOS camera. Complete quantitative phase image reconstruction and image processing were performed in Q-PHASE control software. Cell dry mass values were derived according to {Prescher, 2005 #177} and {Park, 2018 #178} from the phase (eq. (1)), where m is cell dry mass density (in pg/μm2), φ is detected phase (in rad), λ is wavelength in μm (0.65 μm in Q-PHASE), and α is specific refraction increment (≈0.18 μm3/pg). All values in the formula except the Phi are constant. Phi (Phase) is the value measured directly by the microscope. Integrated phase shift through a cell is proportional to its dry mass, which enables studying changes in cell mass distribution (Park et al., 2018).</p>
<p><strong>File description</strong></p>
<p>There are three archives included for particular cell lines:</p>
<ul>
<li>QPI_annotated_timelapse_DU145.zip for DU-145 cells</li>
<li>QPI_annotated_timelapse_PNT1A.zip for PNT1A cells</li>
<li>QPI_annotated_timelapse_LNCaP.zip for LNCaP cells</li>
</ul>
<p>The archive includes of following files:</p>
<ul>
<li><strong>Tiff with time-lapse</strong> quantitative phase image (32-bit files 600x600px with values in pg/um2 with framerate 1 frame/3minutes with 1.59 px/um), named <em>QPI_cellline_treatment_FOV.tiff</em></li>
<li><strong>Tiff file with segmentation</strong> mask for particular cells named <em>mask_cellline_treatment_FOV.tiff</em></li>
<li><strong>xlsx table</strong> with cell death type (1 for apoptosis, 2 for necrosis, 3 for ambiguous/surviving) and time of death for representative cell number from mask, named <em>labels_cellline_treatment_FOV.xlsx</em></li>
</ul>
<p>file naming has following conventions:</p>
<ul>
<li>cell names: DU145, PNT1A, LNCaP for particular cell line</li>
<li>treatments: st, bp, do for staurosporine, black phosphorus and doxorubicin</li>
<li>fields of view: 1 to 7</li>
</ul>
<p>e.g. QPI_DU145_st_4.tif, mask_DU145_st_4.tif, labels_DU145_st_4.xlsx</p>
<p>Note that correlative fluorescence images are available at <a href="https://doi.org/10.5281/zenodo.4531900">10.5281/zenodo.4531900</a></p>This work was supported by funds from the Faculty of Medicine, Masaryk University to Junior researcher (Jan Balvan), and by Grant Agency of the Czech Republic (18–24089 S)
Biocompatible protein cages for encapsulation and internatization of small interfering RNA
No full textThis thesis is focused on creation of apoferritin nanocarrier with encapsulated small interfering RNA marked with fluorescent dye. Main objectives are optimization of pH and amount of siRNA encapsulated into apoferritin cavity and physicochemical characteristics of created nanocarrier. First part deals with theoretical knowledge necessary for understanding concept of this thesis. Second part describes used methods and evaluated results. Created apoferritin nanocarriers were optimal in size with great hemocompatibility, but long-term stability didn’t meet our expectations
Phenotype of melanocytes under physiological and pathological conditions
Get PDFIn addition to the dominant keratinocytes and fibroblasts, melanocytes are also indispensable representatives of skin cell populations. Melanocytes are pigment cells whose primary function is to produce the pigment melanin, which is important for protecting keratinocytes from harmful ultraviolet radiation. Excessive exposure to this radiation is a risk factor for the development of skin tumours, including malignant melanoma of the skin, in which pathological transformation of melanocytes into melanoma cells occurs. The presented thesis focuses on 4 thematic areas associated mainly with malignant melanoma. In the first thematic area, the increasing incidence of malignant skin melanoma is associated with the ageing of the population. One of the reasons seems to be the more frequent occurrence of proinflammatory setting in the ageing organism. It prepares a suitable environment for tumour development. The second thematic area focuses on new approaches that could expand the range of diagnostic methods for the early detection of malignant melanoma. The first approach methodically uses the detection of proinflammatory molecules in the patient's serum. Higher serum levels of IL-6 and IL-8 correlate with an unfavourable patient prognosis. The second approach is based on the possibility of detecting a..
Fluorescence microscopy timelapse dataset of PNT1A, DU-145 and LNCaP cells with annotated caspase 3,7-dependent and independent cell death
No full textTime-lapse dataset of prostatic cell lines (DU-145, PNT1A, LNCaP) exposed to cell death-inducing compounds (staurosporine, doxorubicin) and black phosphorus. Fluorescence dataset of correlative microscopy performed together with quantitative phase microscopy (QPI, dataset available at doi.org/10.5281/zenodo.2601562 together with annotations. The time-lapse dataset is annotated as follows: (1) cell masks and cell numbers, (2) by cell death type and timepoint of death in the attached xlsx file. This dataset is supplementary to the article:
Vicar, T., Raudenska, M., Gumulec, J. et al. The Quantitative-Phase Dynamics of Apoptosis and Lytic Cell Death. Sci Rep 10, 1566 (2020). https://doi.org/10.1038/s41598-020-58474-w
Code is available at https://github.com/tomasvicar/CellDeathDetect
Methods
Cell culture and cultured cell conditions
LNCaP cell line was established from a lymph node metastase of the hormone-refractory patient and contains a mutation in the AR gene. This mutation creates a promiscuous AR that can bind to different types of steroids. LNCaP cells are AR-positive, PSA-positive, PTEN-negative and harbor wild-type p53. PNT1A is immortalized non-tumorigenic epithelial cell line. PNT1A cells harbour wild-type p53. However, SV40 induced T-antigen expression inhibits the activity of p53. This cell line had lost the expression of androgen receptor (AR) and prostate-specific antigen (PSA) (Raudenska, 2019). DU-145 cell line is derived from the metastatic site in the brain and contains P223L and V274F mutations in p53. This cell line is PSA and AR-negative and androgen independent (Chappell, 2012). All cell lines used in this study were purchased from HPA Culture Collections (Salisbury, UK). and were cultured in RPMI-1640 medium with 10 % FBS. The medium was supplemented with antibiotics (penicillin 100 U/ml and streptomycin 0.1 mg/ml). Cells were maintained at 37°C in a humidified (60%) incubator with 5% CO2 (Sanyo, Japan).
Correlative time-lapse quantitative phase-fluorescence imaging
QPI and fluorescence imaging were performed by using multimodal holographic microscope Q-PHASE (TESCAN, Brno, Czech Republic). To determine the amount of caspase-3/7 product accumulation, cells were loaded with 2 µM CellEventTM Caspase-3/7 Green Detection Reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol and visualized using FITC 488 nm filter This channel indicated as GREEN. To detect the cells with a loss of plasma membrane integrity, cells were stained with 1 ug/ml propidium iodide (Sigma Aldrich Co., St. Louis, MO, USA) and visualized using TRITC 542 nm filter (files indicated as RED). Nuclear morphology and chromatin condensation were analyzed using Hoechst 33342 nuclear staining (ENZO, Lausen, Switzerland) and visualized using DAPI 461 nm filter (indicated as BLUE). Cells were cultivated in Flow chambers μ-Slide I Lauer Family (Ibidi, Martinsried, Germany). To maintain standard cultivation conditions (37°C, humidified air (60%) with 5% CO2) during time-lapse experiments, cells were placed in the gas chamber H201 - for Mad City Labs Z100/Z500 piezo Z-stages (Okolab, Ottaviano NA, Italy). To image enough cells in one field of view, lens Nikon Plan 10/0.30 were chosen. For each cell line and each treatment, seven fields of view were observed with the frame rate 3 mins/frame for 24 or 48 h respectively. Holograms were captured by CCD camera (XIMEA MR4021 MC-VELETA), fluorescence images were captured using ANDOR Zyla 5.5 sCMOS camera. Complete quantitative phase image reconstruction and image processing were performed in Q-PHASE control software. Cell dry mass values were derived according to {Prescher, 2005 #177} and {Park, 2018 #178} from the phase (eq. (1)), where m is cell dry mass density (in pg/μm2), φ is detected phase (in rad), λ is wavelength in μm (0.65 μm in Q-PHASE), and α is specific refraction increment (≈0.18 μm3/pg). All values in the formula except the Phi are constant. Phi (Phase) is the value measured directly by the microscope. Integrated phase shift through a cell is proportional to its dry mass, which enables studying changes in cell mass distribution (Park et al., 2018).
File description
There are three archives included for particular cell lines in this dataset:
DU145_fluorescence.zip for timelapse of DU-145 cells
PNT1A_fluorescence.zip for timelapse of PNT1A cells
LNCaP_fluorescence.zip for timelapse of LNCaP cells
These archives include of following files:
BLUE_cellline_treatment_fieldofview.tif Hoechst 33342 nuclear staining (e.g. BLUE_DU145_do_1.tif for DU-145 cells treated with doxorubicin, FOV1)
GREEN_cellline_treatment_fieldofview.tif 2 µM CellEventTM Caspase-3/7 Green Detection Reagent (e.g. GREEN_DU145_do_1.tif )
RED_cellline_treatment_fieldofview.tif 1 ug/ml propidium iodide-stained cells (e.g. RED_DU145_do_1.tif)
(all fluorescent files are 16-bit 600x600px with values in pg/um2 with framerate 1 frame/3minutes with 1.59 px/um)
file naming has following conventions:
cell names: DU145, PNT1A, LNCaP for particular cell line
treatments: st, bp, do for staurosporine, black phosphorus and doxorubicin
fields of view: 1 to 7
Please note that in the second dataset doi.org/10.5281/zenodo.2601562 annotations and quantitative phase image are presentThis work was supported by funds from the Faculty of Medicine, Masaryk University to Junior researcher (Jan Balvan), and by Grant Agency of the Czech Republic (18–24089 S)
Dataset of PNT1A and PC-3 cells - efect of FITC phototoxicity, quantitative phase imaging (1/2)
No full textPart of article Feith, M,Vičar, T., Gumulec, J., Raudenská, M. Wingren, AG, Masařík, M., Balvan, J. Quantitative Phase Dynamics of Cancer Cell Populations Affected by Blue Light, Appl. Sci. 2020, 10
Increased exposition to blue light may induce many changes in cell behavior and significantly affect the critical characteristics of cells. Here we show that multimodal holographic microscopy (MHM) within advanced image analysis is capable of correctly distinguishing between changes in cell motility, cell dry mass, cell density, and cell death induced by blue light. We focused on the effect of blue light with a wavelength of 485 nm on morphological and dynamical parameters of four cell lines, malignant PC-3, A2780, G361 cell lines, and the benign PNT1A cell line. We used MHM with blue light doses 24 mJ/cm2, 208 mJ/cm2 and two kinds of expositions (500 and 1000 ms) to acquire real-time quantitative phase information about cellular parameters. It has been shown that specific doses of the blue light significantly influence cell motility, cell dry mass and cell density. These changes were often specific for the malignant status of tested cells. Blue light dose 208 mJ/cm2 × 1000 ms affected malignant cell motility but did not change the motility of benign cell line PNT1A. This light dose also significantly decreased proliferation activity in all tested cell lines but was not so deleterious for benign cell line PNT1A as for malignant cells. Light dose 208 mJ/cm2 × 1000 ms oppositely affected cell mass in A2780 and PC-3 cells and induced different types of cell death in A2780 and G361 cell lines. Cells obtained the least damage on lower doses of light with shorter time of exposition.
Materials and Methods
Cell Lines
The PC-3, A2780, PNT1A, and G361 cell lines were purchased from HPA Culture Collections (Salisbury, UK). PC-3 prostate cancer cell line was derived from bone metastasis of a 4-grade prostatic adenocarcinoma of a 62-year-old Caucasian male The A2780 cell line was derived from the ovarian carcinoma of a nontreated patient according to ECACC. PNT1A cell line was established from prostatic epithelial tissue of healthy 35-years old male and immortalized by plasmid transfection containing the SV40 genome with defective replication origin. The G361 cell line was established from a malignant melanoma of a 31-year-old male Caucasian. The G361 cells produce melanin for up to 50 population doublings. As the aim of this study is to compare the effect of blue light on the cell lines differing by morphology, transformation state, sensitivity to cell death, and origin, we decided to use the cell lines listed above. PC-3 cells are larger in comparison with small A2780 cells. Benign PNT1A cell line differs from malignant PC-3, and all four cell lines are derived from diverse tissues of origin. Furthermore, melanoma G361 cells expressing melanin may differ in the reaction of cells to blue light exposure.
Cell Cultivation
All four cell lines were cultivated in 25 cm2 flasks with 5 ml of media at 37 °C in a humidified incubator (60%) with 5% CO2 (Sanyo, Osaka City, Japan). Cell lines A2780, PNT1A and G361 were cultured in RPMI-1640 medium with phenol red indicator, L–glutamine, FBS and antibiotics penicillin/streptomycin (Sigma Aldrich Co., St. Louise, MO, USA). For the PC-3 cell line cultivation, Ham´s F-12 medium with FBS and antibiotics (Sigma Aldrich Co., St. Louis, MO, USA) was used. The same supplementation with antibiotics (penicillin 100 U/mL and streptomycin 0.1 mg/mL) and 10% FBS was used in both media. The cell medium was changed two times per week. Cell subculturing was done with 10% of trypsin solution (PAA, Pasching, Austria) with previous washing with EDTA (0.02% in PBS buffer).
QPI and Holographic Microscopy and Fluorescence Setting
QPI was performed by using a Q-PHASE multimodal holographic microscope (Telight, Brno, CZ). The Q-PHASE is equipped with fluorescence module using a halogen lamp as a non-coherent source of blue light. In this work, the module was used as a source of blue light for treatment of observed cell lines. The 485 nm light waves are emitted by the fluorescence light source of the attached module. Before the imaging experiment, cells were cultivated overnight in a concentration of 7000 cells/mL in flow chamber µ-Slide I Lauer Family (Ibidi, Martinsried, Germany). During the measurements, the chamber with cells was incubated in 37 °C humidified, 5% CO2 atmosphere in H201–for Mad City Labs Z100/Z500 piezo Z-stages (Okolab, Ottaviano NA, Italy). Images and holograms were captured with lens Nikon Plan 10/0.3 and CCD camera (XIMEA MR4021 MC-VELETA, Münster, Germany) respectively. The fluorescence mode used was a plasma light source (Sutter Instrument Lambda XL Novato, CA, USA). Cells were irradiated with a 485 nm light with a 25 nm bandwidth. Light doses 0 mJ/cm2, 24 mJ/cm2 and 208 mJ/cm2 were achieved by the combination of time exposition and light intensity.
The images were acquired automatically from seven positions every 3 min for 24 h. Holographic images were collected by custom software and raw data were numerically reconstructed. The numerical reconstruction was performed by custom software where the established methods of the fast Fourier-transform and phase unwrapping are implemented. The output from the software is an unwrapped phase image. This image has high intrinsic contrast and can be processed by an available image processing software. The unwrapped phase image is integrated phase shift through the cell and it is proportional to integrated cell dry mass density
Self-propelled magnetic dendrite-shaped microrobots for photodynamic prostate cancer therapy
No full textPhotocatalytic micromotors that exhibit wireless and controllable motion by light have been extensively explored for cancer treatment by photodynamic therapy (PDT). However, overexpressed glutathione (GSH) in the tumor microenvironment can down-regulate the reactive oxygen species (ROS) level for cancer therapy. Herein, we present dendrite-shaped light-powered hematite microrobots as an effective GSH depletion agent for PDT of prostate cancer cells. These hematite microrobots can display negative phototactic motion under light irradiation and flexible actuation in a defined path controlled by an external magnetic field. Non-contact transportation of micro-sized cells can be achieved by manipulating the microrobot’s motion. In addition, the biocompatible microrobots induce GSH depletion and greatly enhance PDT performance. The proposed dendrite-shaped hematite microrobots contribute to developing dual light/magnetic field-powered micromachines for the biomedical field
Label-Free Nuclear Staining Reconstruction in Quantitative Phase Images Using Deep Learning
No full text
Cisplatin enhances cell stiffness and decreases invasiveness rate in prostate cancer cells by actin accumulation
No full textWe focused on the biomechanical and morphological characteristics of prostate cancer cells and their changes resulting from the effect of docetaxel, cisplatin, and long-term zinc supplementation. Cell population surviving the treatment was characterized as follows: cell stiffness was assessed by atomic force microscopy, cell motility and invasion capacity were determined by colony forming assay, wound healing assay, coherence-controlled holographic microscopy, and real-time cell analysis. Cells of metastatic origin exhibited lower height than cells derived from the primary tumour. Cell dry mass and CAV1 gene expression followed similar trends as cell stiffness. Docetaxel- and cisplatin-surviving cells had higher stiffness, and decreased motility and invasive potential as compared to non-treated cells. This effect was not observed in zinc(II)-treated cells. We presume that cell stiffness changes may represent an important overlooked effect of cisplatin-based anti-cancer drugs. Atomic force microscopy and confocal microscopy data images used in our study are available for download in the Zenodo repository (https://zenodo.org/, Digital Object Identifiers:10.5281/zenodo.1494935).We focused on the biomechanical and morphological characteristics of prostate cancer cells and their changes resulting from the effect of docetaxel, cisplatin, and long-term zinc supplementation. Cell population surviving the treatment was characterized as follows: cell stiffness was assessed by atomic force microscopy, cell motility and invasion capacity were determined by colony forming assay, wound healing assay, coherence-controlled holographic microscopy, and real-time cell analysis. Cells of metastatic origin exhibited lower height than cells derived from the primary tumour. Cell dry mass and CAV1 gene expression followed similar trends as cell stiffness. Docetaxel- and cisplatin-surviving cells had higher stiffness, and decreased motility and invasive potential as compared to non-treated cells. This effect was not observed in zinc(II)-treated cells. We presume that cell stiffness changes may represent an important overlooked effect of cisplatin-based anti-cancer drugs. Atomic force microscopy and confocal microscopy data images used in our study are available for download in the Zenodo repository (https://zenodo.org/, Digital Object Identifiers:10.5281/zenodo.1494935)
Cisplatin enhances cell stiffness and decreases invasiveness rate in prostate cancer cells by actin accumulation: dataset of confocal and atomic force microscopy
No full text<p><strong>Summary</strong></p>
<p>Dataset of imaging data of the experiment "Cisplatin enhances cell stiffness: Biomechanical profiling of prostate cancer cells". This dataset includes image data of <em>atomic force microcopy</em> (Young modulus) and <em>confocal microscopy</em>(staining of F-actin and β-tubulin) of prostate cell lines PNT1A, 22Rv1, and PC-3. </p>
<p><strong>Materials and Methods</strong></p>
<p><em>Cells, cell culture conditions</em></p>
<p>Cells confluent up to 50–60% were washed with a FBS-free medium and treated with a fresh medium with FBS and required antineoplastic drug concentration (IC50 concentration for the particular cell line). The cells were treated with 93 µM (PC-3), 38 µM (PNT1A), and 24 µM (22Rv1) of cisplatin (Sigma-Aldrich, St. Louis, Missouri), respectively. IC50 concentrations used for treatment with docetaxel (Sigma-Aldrich, St. Louis, Missouri) were 200nM for PC-3, 70nM for PNT1A, and 150nM for 22Rv1. </p>
<p><em>Long-term zinc (II) treatment of cell cultures</em></p>
<p>Cells were cultivated in the constant presence of zinc(II) ions. Concentrations of zinc(II) sulphate in the medium were increased gradually by small changes of 25 or 50 µM. The cells were cultivated at each concentration no less than one week before harvesting and their viability was checked before adding more zinc. This process was used to select zinc resistant cells naturally and to ensure better accumulation of zinc within the cells (accumulation of zinc is usually poor during the short-term treatment of prostate cancer cells). Total time of the cultivation of cell lines in the zinc(II)-containing media exceeded one year. Resulting concentrations of zinc(II) in the media (IC50 for the particular cell line) were 50 µM for the PC-3 cell line, 150 µM for the PNT1A cell line, and 400 µM for the 22Rv1 cell line. The concentrations of zinc(II) in the media and FBS were taken into account. </p>
<p><em>Actin and tubulin staining</em></p>
<p>β-tubulin was labeled with anti- β tubulin antibody [EPR1330] (ab108342) at a working dilution of 1/300. The secondary antibody used was Alexa Fluor® 555 donkey anti-rabbit (ab150074) at a dilution of 1/1000. Actin was labeled with Alexa Fluor™ 488 Phalloidin (A12379, Invitrogen); 1 unit per slide. For mounting Duolink® In Situ Mounting Medium with DAPI (DUO82040) was used. The cells were fixed in 3.7% paraformaldehyde and permeabilized using 0.1% Triton X-100. </p>
<p><em>Confocal microscopy</em></p>
<p>The microscopy of samples was performed at the Institute of Biophysics, Czech Academy of Sciences, Brno, Czech Republic. Leica DM RXA microscope (equipped with DMSTC motorized stage, Piezzo z-movement, MicroMax CCD camera, CSU-10 confocal unit and 488, 562, and 714 nm laser diodes with AOTF) was used for acquiring detailed cell images (100× oil immersion Plan Fluotar lens, NA 1.3). Total 50 Z slices was captured with Z step size 0.3 μm.</p>
<p><em>Atomic force microscopy</em></p>
<p>We used the bioAFM microscope JPK NanoWizard 3 (JPK, Berlin, Germany) placed on the inverted optical microscope Olympus IX‑81 (Olympus, Tokyo, Japan) equipped with the fluorescence and confocal module, thus allowing a combined experiment (AFM‑optical combined images). The maximal scanning range of the AFM microscope in X‑Y‑Z range was 100‑100‑15 µm. The typical approach/retract settings were identical with a 15 μm extend/retract length, Setpoint value of 1 nN, a pixel rate of 2048 Hz and a speed of 30 µm/s. The system operated under closed-loop control. After reaching the selected contact force, the cantilever was retracted. The retraction length of 15 μm was sufficient to overcome any adhesion between the tip and the sample and to make sure that the cantilever had been completely retracted from the sample surface. Force‑distance (FD) curve was recorded at each point of the cantilever approach/retract movement. AFM measurements were obtained at 37°C (Petri dish heater, JPK) with force measurements recorded at a pulling speed of 30 µm/s (extension time 0.5 sec).</p>
<p>The Young's modulus (E) was calculated by fitting the Hertzian‑Sneddon model on the FD curves measured as force maps (64x64 points) of the region containing either a single cell or multiple cells. JPK data evaluation software was used for the batch processing of measured data. The adjustment of the cantilever position above the sample was carried out under the microscope by controlling the position of the AFM‑head by motorized stage equipped with Petri dish heater (JPK) allowing precise positioning of the sample together with a constant elevated temperature of the sample for the whole period of the experiment. Soft uncoated AFM probes HYDRA-2R-100N (Applied NanoStructures, Mountain View, CA, USA), i.e. silicon nitride cantilevers with silicon tips are used for stiffness studies because they are maximally gentle to living cells (not causing mechanical stimulation). Moreover, as compared with coated cantilevers, these probes are very stable under elevated temperatures in liquids – thus allowing long-time measurements without nonspecific changes in the measured signal.</p>
<p><strong>Identification of files</strong></p>
<p>Files are separated into individual zip files. The dataset of <em>confocal microscopy </em>is separated based on treatments: untreated control, docetaxel-treated cells, cisplatin-treated cells, zinc-treated cells. Filenames actin_tubulin_Zstack_cisplatin.zip, actin_tubulin_Zstack_untreated_control.zip, actin_tubulin_Zstack_zinc.zip, actin_tubulin_Zstack_docetaxel.zip. Files included in these ZIP archives are named as follows: "cellline_treatment_FOV". Files are 3-layer 16bit tiff files with layer sequence as follows: F-Actin (Phalloidin)/b-tubulin/Hoechst 33342. The dataset contains 242 FOVs of three cell line types/three treatments + one control, files are Z-stacks made of 50 slices.</p>
<p>The dataset of <em>atomic force microscopy </em>(AFM) is included in one ZIP archive "AFM_YoungModulus_SetpointHeight.zip", which includes data on Young modulus and Setpoint Height of cell lines 22Rv1, PNT1A and PC-3 and treatments zinc, docetaxel, cisplatin (+control), i.e. identical like for confocal microscopy. The file naming is as follows: "AFM_cellline_treatment_FOV_Youngmodulus.tif" for Young modulus and "AFM_cellline_treatment_FOV_setpointheight.tif" for setpoint height. The data are filtered 32-bit tiff images, where the pixel value correspond to cell stiffness (young modulus) in Pa or setpoint height in m.</p>This work was supported by funds from the Faculty of Medicine, Masaryk University to Junior researcher (Martina Raudenska), by Grant Agency of the Czech Republic (18-24089S) and by CIISB research infrastructure project LM2015043 funded by MEYS CR (support to AFM measurements). We thank Dr. Martin Falk from the Institute of Biophysics, Czech Academy of Sciences, Brno, Czech Republic for the performance of confocal microscopy
- …
