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.
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.
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.
| Compound | Cytotoxicity | Live | Apoptotic | Necrotic | |||
|---|---|---|---|---|---|---|---|
| MEC | MR(%) | MEC | MR(%) | MEC | MR(%) | ||
| 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).
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.
Learn More
Learn more about toxicology in our popular Mechanisms of Drug-Induced Toxicity guide
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