DOI: 10.5061/dryad.9s4mw6mrs
title: Fluorescence time-lapse images of MDA-MB-231 cells expressing FUCCI(CA)2 (Part 2/2)

description: Understanding the details of the cell cycle at the level of individual
 cells is critical for both cellular biology and cancer research. While
 existing methods using specific fluorescent markers have advanced our
 ability to study the cell cycle in cells that adhere to surfaces, there is
 a clear gap when it comes to non-adherent cells. In this study, we combine
 a specialized surface to improve cell attachment, the genetically-encoded
 FUCCI(CA)2 sensor, an automated image processing and analysis pipeline,
 and a custom machine-learning algorithm. This combined approach allowed us
 to precisely measure the duration of different cell cycle phases in
 non-adherent cells. Our method provided detailed information from hundreds
 of cells under different experimental conditions in a fully automated
 manner. We validated this approach in two different Acute Myeloid Leukemia
 (AML) cell lines, NB4 and Kasumi-1, which have unique cell cycle
 characteristics. Additionally, we tested the impact of drugs affecting the
 cell cycle in NB4 cells. Importantly, our cell cycle analysis system is
 freely available and has also been validated for use with adherent cells.
 In summary, this report introduces a comprehensive, automated method for
 studying the cell cycle in both adherent and non-adherent cells, offering
 a valuable tool for cancer research and drug development.
description: Images were acquired with a Leica Thunder Imager (Leica
 Microsystems, Wetzlar, Germany), equipped with a Lumencor Spectra X Light
 Engine (Lumencor, Beaverton, USA) for fluorescence excitation, a motorized
 stage and a Leica DFC9000 GTC camera. For non-adherent cells, images were
 acquired with LAS X software (Leica Microsystems, Wetzlar, Germany,
 version 3.7.5.24914) using a 10X/0.32NA dry objective. The mCherry and
 mVenus signals were detected respectively with 540-580 nm and 460-500 nm
 excitation filters, 585 and 505 nm dichroic mirrors and 592-668 nm and
 512-542 nm emission filters. The brightfield channel was also acquired for
 representation purposes. We imaged 10 fields of views per well and focal
 points were manually set in each position before starting the acquisition
 and kept constant during the whole time-lapse thanks to the Adaptive Focus
 Control (AFC, Leica Microsystems). The total duration of the time-lapse on
 dherent cells was 120 hours, and the time interval was set to 0.5
 hour.
description: # FUCCI(CA)2\_imaging\_adherent\_cells
 [https://doi.org/10.5061/dryad.9s4mw6mrs](https://doi.org/10.5061/dryad.9s4mw6mrs) ## Description of the data and file structure One hundred thousand MDA-MB-231 cells expressing the FUCCI(CA)2 probe were plated in a Tethis SBS 12-well multi-well plate. Images were acquired with a Leica Thunder Imager (Leica Microsystems, Wetzlar, Germany). The mCherry and mVenus signals were detected respectively with 540-580 nm and 460-500 nm excitation filters, 585 and 505 nm dichroic mirrors and 592-668 nm and 512-542 nm emission filters. The brightfield channel was also acquired. Channel 1: BF Channel 2: mVenus Channel 3: mCherry Pixel size: 650 nm Z-step: 5.3 um time-frame: 30 min total duration: 120h ### Files and variables Compressed .lif files. #### **File:** FUCCI\_adherent\_cells\_B1.zip, FUCCI\_adherent\_cells\_B2.zip **Description:** compressed .lif files containing 10 regions (R1-R10), 3 channels (and BF, green and red ), 2 Z planes and 241 time frames. FUCCI_adherent_cells_B1 refers to the well B1, FUCCI_adherent_cells_B2 refers to the well B2. ## Code/software This files can be opened, visualized and analysed with Fiji/ImageJ, after extraction or the LasX Leica software