Analysis of Cellular Senescence

Introduction

Cellular senescence is the phenomenon in which cell division is irreversibly arrested upon reaching a division limit following repeated divisions. Cells in this state are referred to as senescent cells. Cellular senescence is caused by genome instability resulting from telomere shortening or accumulation of DNA damage. It is believed that this arresting of division inhibits the canceration of cells. On the other hand, recent research has shown that senescent cells secrete cytokines, growth factors and matrix metalloproteinase through the SASP (Senescence-Associated Secretory Phenotype) phenomenon, inducing the canceration of surrounding cells. Because of this, cell senescence has attracted a lot of attention in recent years, including also its relation to oncogenesis and individual aging. This application note shows an example of cellular senescence evaluation. A senescent cell detection kit (Cellular Senescence Detection Kit -SPiDER-βGal, Dojindo Laboratories Co., Ltd.) that indexes the activity of the senescent cell marker SA-β-gal (Senescence-Associated β-galactosidase) was used. Imaging was performed using the CQ1 and analysis was carried out using the CellPathfiner high content analysis software.

1.Analysis via living cell SPiDER-βGal staining

Experiment Procedure

1. WI-38 cells with passage number 0 and 13 were inoculated in 96-well plates (Greiner #655896) and incubated for 24 hours.
2. SA-β-gal (senescent cell marker) was stained according to the Cellular Senescence Detection Kit -SPiDER-βGal protocol.
3. Nuclear staining was carried out concurrently (Hoechst33342).
4. The cells were washed with HBSS then imaged with the CQ1. The imaging conditions were as follows: 10x objective lens, two wavelengths: 405nm (Hoechst33342) and 488nm (SPiDER-β-gal), 8 scopes per well.
5. Image analysis was performed using CellPathfinder.

Fig. 1: Detection of senescent cells using SA-β-gal staining
Cells with passage number 0 (A) and passage number 13 (B)
Blue: Hoechst33342, Green: SPiDER-βGal (C) and (D) are the analysis results for (A) and (B), respectively.
Nuclear regions were recognized using 405nm imaging and cytoplasm regions were recognized using 488nm imaging. Cells with average intensity above a certain level in cytoplasm regions were identified (red outline) as SA-β-gal positive cells (senescent cells).
(E) Senescent cell ratio (%) across all nuclei. The ratio of SA-β-gal positive cells was approximately six times higher in the passage number 13 cells (orange) than the passage number 0 cells (blue).
(F) Total intensity histogram for SA-β-gal in cytoplasm. There were a larger number of cells with high total intensity among the passage number 13 cells (orange) than the passage number 0 cells (blue).

2.Analysis via fixed cell SPiDER-βGal and co-staining of the DNA damage marker γH2AX

Experiment Procedure

1. WI-38 cells with passage number 1 and 10 were inoculated in 96-well plates and incubated for 24 hours.
2. SA-β-gal (senescent cell marker) was stained according to the Cellular Senescence Detection Kit -SPiDER-βGal protocol.
3. The cells were fixed (4% PFA) and membrane permeation was performed (0.1% Triton).
4. γH2AX (DNA damage marker) was stained using anti-γH2AX antibody (CST Japan #2577S) and Alexa Fluor 647 secondary antibody.
5. Nuclear staining was carried out concurrently using DAPI.
6. The cells were imaged with the CQ1. The imaging conditions were as follows: 10x objective lens, three wavelengths: 405nm (DAPI), 488nm (SPiDER-β-gal) and 640nm (γH2AX), 6 scopes per well.
7. Image analysis was performed using CellPathfinder.

Fig. 2: SA-β-gal and γH2AX co-staining
Cells with passage number 1 (A) and passage number 10 (B)
Blue: DAPI, Green: SPiDER-βGal, Pink: γH2AX
(C) and (D) are the analysis results for (A) and (B), respectively. Nuclear regions (blue outline) were recognized using 405nm imaging, intracellular dots (yellow) were recognized using 640nm imaging, and cytoplasm regions were recognized using 488nm imaging. Cells with average intensity above a certain level in cytoplasm regions were identified (orange outline) as SA-β-gal positive cells (senescent cells).
(E) 40x objective lens image of passage number 10 cells. Intranuclear γH2AX localization is clearly visible.
(F) Senescent cell ratio (%) across all nuclei
(G) Histogram of the ratio γH2AX area / nuclear area. Light blue: SA-β-gal negative cells, Red: SA-β-gal positive cells. The count of SA-β-gal positive cells with high Ratio_γH2AX/NucArea values was larger for the passage number 10 cells (right) than passage number 1 (left).

Results and Discussion

It was confirmed that live cell autophagy can be easily observed using the CQ1 and DALGreen-Autophagy Detection. Also, autophagy was induced in the media not containing amino acid and inhibited by adding Bafilomycin. The CQ1 allows for the observation of changes with time while maintaining the culture environment through the use of its stage heater for the control of temperature and humidity, as well as the concentrations of CO2 and O2 through the combined use of a gas mixer.

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