DOI: 10.5281/zenodo.4531899
title: Fluorescence microscopy timelapse dataset of PNT1A, DU-145 and LNCaP cells with annotated caspase 3,7-dependent and independent cell death

description: Time-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. <em>et al.</em> The Quantitative-Phase Dynamics of Apoptosis and Lytic Cell Death. <em>Sci Rep</em> <strong>10, </strong>1566 (2020). https://doi.org/10.1038/s41598-020-58474-w Code is available at https://github.com/tomasvicar/CellDeathDetect <strong>Methods</strong> <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. 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). <em>Correlative time-lapse quantitative phase-fluorescence imaging</em> 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). <strong>File description</strong> 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: <strong>BLUE_cellline_treatment_fieldofview.tif </strong>Hoechst 33342 nuclear staining (<em>e.g.<strong> </strong>BLUE_DU145_do_1.tif for DU-145 cells treated with doxorubicin, FOV1)</em> <strong>GREEN_cellline_treatment_fieldofview.tif </strong>2 µM CellEventTM Caspase-3/7 Green Detection Reagent <em>(e.g. GREEN_DU145_do_1.tif )</em> <strong>RED_cellline_treatment_fieldofview.tif </strong>1 ug/ml propidium iodide-stained cells<strong> </strong><em>(e.g. RED_DU145_do_1.tif)</em> (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 present
description: 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).