This site uses cookies. By continuing to browse the site you are agreeing to our use of cookies. Find out more here.
ADME PK

Apoptosis and Necrosis Assay
(Flow Cytometry)

Detect compound induced cell death using Cyprotex’s flow cytometric apoptosis and necrosis assay.

Cyprotex deliver consistent, high quality data with the flexibility to adapt protocols based on specific customer requirements.

Background Information

  • Flow cytometry allows analysis of multiple parameters of cell health and provides a high throughput analysis of compound cytotoxicity.
  • Apoptosis and necrosis occur during cell death in response to cytotoxic conditions. Cyprotex’s apoptosis and necrosis assay utilises Annexin V and propidium iodide dual staining to monitor cellular death.
  • Annexin V-FITC binds to phosphatidylserine which is translocated from the inner to outer plasma membrane during early apoptosis. Propidium Iodide is a cell impermeable nuclear dye which is excluded by viable and early apoptotic cells. However, it is taken up by necrotic or late apoptotic cells resulting in red fluorescence.
  • A dose dependent increase in apoptotic or necrotic cells, accompanied by a decrease in live cells, indicates a compound is inducing cellular death.
Increased research on the mechanisms of cell death in recent years has led to the understanding that apoptosis and necrosis involve different cellular pathways and that these differences can have important implications when considering overall mechanisms of toxicity.

1Elmore SA et al., (2016) Recommendations from the INHAND Apoptosis/Necrosis Working Group. Toxicologic Pathology 44(2); 173-188

Protocol

Protocol for Apoptosis and Necrosis Assay

Cell Type TK6, CHO-K1 (other lines available on request)
Analysis Platform Intellicyte iQue Screener PLUS
Test Article Requirements 5-10 mg solid or equivalent solution
Time Points Dosing time 24 hours (other time points available on request)
Quality Control Assay appropriate control
Endpoints Quantification of live, dead and early apoptotic cells.

Data

Data from Cyprotex's Apoptosis and Necrosis Assay

 
 
Figure 1
Flow cytometric analysis of TK6 cells.

TK6 cells were treated with etoposide for 24 hours prior to flow cytometric analysis using the apoptosis and necrosis assay. Live, dead and apoptotic populations were identified by plotting Annexin V intensity against PI intensity.
 
 
 
Figure 2
Graphical representation of flow cytometry data.

TK6 cells were treated with etoposide for 24 hours followed by analysis using the apoptosis and necrosis assay. The percentage of apoptotic and dead cells increases in a dose dependent manner while the live cell percentage decrease proportionally.
CompoundCytotoxicityLiveApoptoticNecrotic
MECMR(%)MECMR(%)MECMR(%)
Rotenone cytotoxic 2.18 41.1 1.21 15.8 <0.04 51.5
Cyclosporine A cytotoxic 1.88 32.5 0.707 54.2 0.868 49.2
Etoposide cytotoxic 0.538 37.4 0.131 12.5 0.0417 50.1
Sunitinib cytotoxic 13.7 9.7 45.8 30 7.38 84.6
Imatinib cytotoxic 62.7 17.5 0.436 41.5 24.4 75.9
Carbonyl cyanide 3-chlorophenylhydrazone cytotoxic 4.33 14.6 2.36 52.7 2.03 73.6
Chlorpromazine cytotoxic 7.07 25.4 3.72 53.2 9.07 54.1
Chlorambucil cytotoxic 9.61 14.1 NR - 0.402 66.9
Amoxicillin non-cytotoxic NR - NR - NR -
Alendronate non-cytotoxic NR - NR - NR -
Table 1

Summary of validation data.

MEC- minimal effective concentration; MR – maximum observed response (% of total cells).

References

1 Elmore SA et al., (2016) Recommendations from the INHAND Apoptosis/Necrosis Working Group. Toxicologic Pathology 44(2); 173-188.

Request a Quote
Speak to a Scientist
Product sheet ADME-Tox Guides Pricing & Discounts

Contact us to discuss your ADME Tox issues or request a quote

Telephone:
North America (East Coast): 888-297-7683
Europe: +44 1625 505100

 

or fill out the form below:

Please give details of the assays you are interested in. Where appropriate please specify one or more species (human, rat, mouse etc.), isoforms (CYP1A1,CYP1B1, etc) or other relevant details.

By clicking on 'Send' I agree that my data is being transferred and used by Cyprotex. For further details please see our privacy policy.

If you would like to be kept informed of our latest services, new research, events and other news please tick the box below:

Close