Apoptosis Assay

Apoptosis Assay2019-06-07T14:02:46-05:00

Background

  • Apoptosis is a form of programmed cell death that is highly regulated within the cell. Apoptosis is often dysregulated in diseases, especially cancer, interrupting the innate ability of cells possessing potentially harmful mutations to be terminated [1].
  • The assessment of apoptosis in 3D cell models or cell monolayers involves measuring the presence or activity of Caspase-3 and/or Caspase-7. Caspases are protease enzymes that play an essential role in apoptosis. During apoptosis, Caspase-3 is activated. which in turn cleaves and activates Caspase-6 and 7 [2].
  • Apoptosis can be assessed by using fluorescent probes for Caspase-3 or Caspase-7, by immunolabeling for Caspase-3/7, or alternatively using other assay formats such as Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL).
  • The assessment of the induction of apoptosis is an important endpoint in the evaluation of the effectiveness of antineoplastic agents on cancer cells.
  • Detection and quantification of apoptotic cells can be accomplished utilizing High Content Screening to image fluorogenic molecular probes which are permeable to cell membranes and are substrates to Caspase-3.
  • In conjunction with other assay endpoints, such as the assessment of cell proliferation or cell viability, the measurement of apoptosis is an useful tool for interrogating the effect of cancer drugs in vitro.
  • 3D cell culture models effectively recapitulate the in vivo tumor environment, and are an effective tool for the evaluation of apoptosis-inducing effects of promising anti-cancer drug compounds.
  • Assessment of apoptosis is critical for the evaluation of the safety and toxicity of test compounds. Assaying the apoptosis-induction of compounds in 3D hepatocellular models is an excellent method for identifying and eliminating lead compounds that possess toxic liabilities early in the drug discovery process.
  • This assay can be coupled with up to two other imaging-based endpoints (e.g. cell proliferation and cell viability), or with immunolabeling to identify the type of cells which become apoptotic from treatments.

Protocol

InstrumentThermoFisher CX7 LZR
Analysis MethodHigh content screening
MarkersNucView fixable apoptosis probe (Biotium)
DAPI (total cell count)
Other apoptosis probes available on request
Cell Model Type3D cell models (e.g. tumor spheroids, organoids, etc.)
or adherent monolayer / cell suspension
Cell Types AvailableATCC cancer cell lines (e.g. A549, BT549, MCF-7)
Primary cells
HepG2, HepaRG, primary human hepatocytes
Client provided cells
Custom models available on request
Test Article Concentration8 point assay (0.05, 0.1, 0.5, 1, 5, 10, 50, 100 µM)
(custom concentrations available)
Single point assay
Number of Replicates3 replicates per concentration
Quality Controls0.5% DMSO (vehicle control)
Doxorubicin (positive control)
Test Article Requirements50 uL of 20 mM solution or equivalent amount of solid
Data DeliveryDose response curves, EC50 values, total cell counts, percent of apoptotic cells for each test concentration
Evaluation of statistical significance of results with respect to vehicle and positive control

General Procedure

  1. 3D cell model generated and grown to approximately 200 μm in diameter. Adherent monolayers grown to confluence.
  2. Treatment with test compounds
  3. After 24 hours, cells are labeled with fixable apoptosis probe.
  4. Nuclei are labeled with DAPI
  5. For 3D models, tissue clearing is applied to render 3D cell models transparent
  6. High content imaging is conducted on well plates
  7. Images are analyzed to quantify total number of cells and total number of apoptotic cells

Data

Figure 1. Maximum Z-projection of Z-stack obtained from High Content Imaging of Wood cell tumor spheroids treated with fulvestrant. Data was quantified by automated image analysis to count total number of cells, and number of apoptotic cells

Figure 2. Representative data showing the observed fraction of apoptotic cells detected in Wood cell tumor spheroids treated with various antineoplastic drugs


References

  1. Green, Douglas (2011). Means to an End: Apoptosis and other Cell Death Mechanisms. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
  2. Wyllie AH (1997). “Apoptosis: an overview”. British Medical Bulletin53 (3): 451–65. doi:10.1093/oxfordjournals.bmb.a011623
This website uses cookies to enhance the user experience. Ok