Membrane integrity-based viability assay
The Invitrogen LIVE/DEAD fixable dead cell stains distinguish between live and dead cells in flow cytometry. The dyes covalently bind to intracellular and extracellular amines, allowing the staining pattern to be preserved following formaldehyde fixation. Choose from eight different fluorescent colors.
This protocol can be used for:
- Identifying live and dead cells using a flow cytometer
This protocol should not be used for:
- Fluorescence microscopy
You will need the following for this protocol:
- Cells growing in culture
- One of the LIVE/DEAD fixable dead cell stains (e.g. LIVE/DEAD Fixable Green Dead Cell Stain Kit (Cat. No. L23101))
- Flow cytometer
Protocol
 1. Thaw vial of dye |
 2. Dilute LIVE/DEAD fixable dead cell stain by adding 50 µL DMSO to vial |
 3. Add 1 mL of cells to a flow cytometer tube in protein-free buffer |
| 4. Add 1 µL of diluted stain to cells |
 5. Mix cells and stain |
 6. Incubate 30 minutes |
 7. Wash cells |
 8. Analyze on flow cytometer |
Protocol tips
- Cell concentration should be 1x104-1x106 cells per mL
- Washing is optional after staining
- Cell staining is preserved after fixation, but fixation is not required
- Protein concentration in buffer should be <1%
Live and dead cells distinguished by flow cytometry using the LIVE/DEAD Fixable Violet Dead Cell Stain Kit.
Emission specifications
| LIVE/DEAD dye | Excitation source | Ex* | Em* |
|---|---|---|---|
| LIVE/DEAD Fixable Blue stain | UV | 350 | 450 |
| LIVE/DEAD Fixable Violet stain | 405 | 416 | 451 |
| LIVE/DEAD Fixable Lime stain | 405 | 405 | 506 |
| LIVE/DEAD Fixable Aqua stain | 405 | 367 | 526 |
| LIVE/DEAD Fixable Yellow stain | 405 | 400 | 575 |
| LIVE/DEAD Fixable Green stain | 488 | 495 | 520 |
| LIVE/DEAD Fixable Olive stain | 488 | 480 | 557 |
| LIVE/DEAD Fixable Orange stain | 561 | 580 | 602 |
| LIVE/DEAD Fixable Red stain | 488 | 595 | 615 |
| 561 | |||
| LIVE/DEAD Fixable Far Red stain | 633/635 | 650 | 665 |
| LIVE/DEAD Fixable Scarlet stain | 633/635 | 700 | 775 |
| LIVE/DEAD Fixable Near-IR (775) stain | 633/635 | 750 | 775 |
| LIVE/DEAD Fixable Near-IR (780) stain | 633/635 | 750 | 780 |
| LIVE/DEAD Fixable Near IR (876) stain | 808 | 840 | 876 |
| *Approximate fluorescence excitation (Ex) and emission (Em) maxima, in nm. | |||