Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 May 5;8(9):e2835.
doi: 10.21769/BioProtoc.2835.

An Image-based Assay for High-throughput Analysis of Cell Proliferation and Cell Death of Adherent Cells

Paula Szalai  1 , Nikolai Engedal  1
Affiliations

An Image-based Assay for High-throughput Analysis of Cell Proliferation and Cell Death of Adherent Cells

Paula Szalai et al. Bio Protoc. .

Abstract

In this protocol, we describe a method to monitor cell proliferation and death by live-cell imaging of propidium iodide (PI)-stained adherent mammalian cells. PI is widely used to assess cell death. However, it is usually used in end-point assays. Recently, we implemented the use of PI for real-time cell death assessment by automated imaging. Cells are seeded in a 96-well format, and after attachment, the treatments are added directly to the wells together with PI. Thereafter, cells are subjected to automated time-lapse imaging and quantification by computer software. Combined analyses of phase-contrast and fluorescence images allow assessment of treatment effects on cell proliferation as well as the extent and kinetics of cell death.

Keywords: Cell death; Cell proliferation; Live-cell imaging; Propidium iodide; Thapsigargin; Time-lapse imaging.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.. Overview of the procedure to monitor cell death in real time using propidium iodide and the IncuCyte ZOOM instrument
Figure 2.
Figure 2.. Analysis of cell confluence and cell death using the IncuCyte ZOOM system.
LNCaP (left) or PC3 (right) cells were treated with DMSO or increasing concentrations of thapsigargin, as indicated, in the presence of 2.5 µg/ml PI. A and B. Cell confluence (the proportion of the surface covered by cells, as determined by phase-contrast images) of LNCaP and PC3 cells plotted over time as absolute percentage (A) or relative to start confluence (B). C. The ratio of red fluorescent (PI-positive) to total cell confluence plotted over time. Means ± SEM of 3 (LNCaP) or 6 (PC3) independent experiments.
Figure 3.
Figure 3.. Examples of live-cell images using the IncuCyte ZOOM system.
Overlayed phase-contrast and fluorescence images (10x magnification) of LNCaP (A) and PC3 (B) cells after 0 h, 24 h, 48 h and 72 h of treatment with DMSO or thapsigargin (Tg), in the presence of PI. Scale bar represents 300 µm.
Figure 4.
Figure 4.. Direct comparison of PI-based cell death analysis using the IncuCyte ZOOM or Flow Cytometry.
PC3 cells were treated for 42 h with DMSO or increasing concentrations of thapsigargin, as indicated, in the presence of PI. Cell death was first analyzed by live-cell imaging using the IncuCyte ZOOM (A), and subsequently by flow cytometry (using BD Bioscience FACSCanto II and the Flowing Software by Perttu Terho) from the same wells (B). For flow cytometry, the cells were harvested with 0.25% Trypsin-EDTA (1x), and 10,000 events were acquired and analyzed as previously described ( Crowley et al., 2016 ). Cellular debris were excluded by gating forward/side scatter plots, and PI-positive cells were gated based on forward scatter/PI fluorescence plots, as described ( Crowley et al., 2016 ). Shown are ratios of red fluorescence to total cell confluence (A) and percentages of gated PI-positive cells (B). Means ± SEM of 3 independent experiments. EC50 values of the maximal thapsigargin effect (with 95% Confidence Intervals) are shown.
Figure 5.
Figure 5.. Examples of confluence mask adjustment using the IncuCyte ZOOM system.
Screenshots of the IncuCyte ZOOM software Analyzer tool for confluence mask (yellow outlines). A. Appropriate adjustment for PC3 cells with segmentation adjustment of 1.8 towards cells (red rectangle) and Filter for areas smaller than 500 µm2 (blue rectangle). B. Example of inappropriate adjustment for PC3 cells with segmentation adjustment of 0.2 towards background (red rectangle) and no Filter (blue rectangle). Marked with arrows are examples of cells/parts of cells which are not included in the confluence mask (red arrows) and debris which are not filtered out (blue arrows). Due to incorrect segmentation adjustment the calculated confluence is too low (9% instead of 39%).
See this image and copyright information in PMC

References

    1. Crowley L. C., Scott A. P., Marfell B. J., Boughaba J. A., Chojnowski G. and Waterhouse N. J.(2016). Measuring cell death by propidium iodide uptake and flow cytometry. Cold Spring Harb Protoc 7: pdb.prot087163. - PubMed
    1. Sehgal P., Szalai P., Olesen C., Praetorius H. A., Nissen P., Christensen S. B., Engedal N. and Moller J. V.(2017). Inhibition of the sarco/endoplasmic reticulum(ER) Ca2+-ATPase by thapsigargin analogs induces cell death via ER Ca2+ depletion and the unfolded protein response . J Biol Chem 292(48): 19656-19673. - PMC - PubMed
    1. Wlodkowic D., Skommer J., McGuinness D., Faley S., Kolch W., Darzynkiewicz Z. and Cooper J. M.(2009). Chip-based dynamic real-time quantification of drug-induced cytotoxicity in human tumor cells. Anal Chem 81(16): 6952-6959. - PMC - PubMed
    1. Zhao H., Oczos J., Janowski P., Trembecka D., Dobrucki J., Darzynkiewicz Z. and Wlodkowic D.(2010). Rationale for the real-time and dynamic cell death assays using propidium iodide. Cytometry A 77(4): 399-405. - PMC - PubMed