Analysis of Cellular Senescence

下载 (520 KB)

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.

Detection of senescent cells using SA-β-gal staining

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.

SA-β-gal and γH2AX co-staining

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.


Our Social Medias

We post our information to the following SNSs. Please follow us.

  Follow us Share our application
•Twitter @Yokogawa_LS Share on Twitter
•Facebook Yokogawa Life Science Share on Facebook
•LinkedIn Yokogawa Life Science Share on LinkedIn

 

Yokogawa's Official Social Media Account List

Social Media Account List


相关产品&解决方案

  • CellPathfinder

    CellPathfinder is designed for our HCA systems, CQ1 and the CellVoyager series. From beginners to experts, the analysis software lets you quantify subtle physiological changes and even label-free samples with various graph options.

    更多
  • CV8000高内涵筛选系统

    CellVoyager CV8000是先进的高内涵筛选系统。改进的内置培养箱可以让客户分析长期的活细胞反应。凭借其可扩展性、4个摄像机、5个激光器和可选的内置移液器,该系统允许日益复杂的分析开发和高内涵筛选。

    更多
  • 生命科学

    横河电机的高含量分析系统和双转盘共聚焦技术提供了高速、高分辨率的活细胞成像,引导了全球行业前沿的研究。

    更多
  • 高内涵分析

    我们的高内涵分析系统利用强大的分析软件解决从基础科学到药物发现筛选的一系列研究应用。

    更多

置顶
WeChat QR Code
横河电机(中国)有限公司