1. Dilute semen sample in Live Cell Imaging Solution plus 10% BSA
Two-color assay indicates live and dead sperm cells
This kit enables analysis of sperm viability and fertilizing potential. SYBR 14 labels live sperm with green fluorescence, and propidium iodide labels membrane-compromised or dead sperm with red fluorescence.
This protocol can be used for:
- Identifying live and dead sperm using a flow cytometer
This protocol should not be used for:
- Fluorescence microscopy. For a fluorescence microscopy protocol, see LIVE/DEAD Sperm Viability Kit Imaging Protocol
You will need the following for this protocol:
Assay protocol
2. Add 900 µL DMSO to SYBR 14 dye to make a stock solution
3. Add 1 µL of SYBR 14 stock solution and 5 µL of propidium iodide solution to 1 mL of diluted semen
4. Incubate for 5–10 minutes at 37°C
5. Analyze on a flow cytometer
Spectral information and storage
| SYBR 14 | Propidium iodide | |
|---|---|---|
| Excitation/Emission | 488/516 nm | 535/617 nm |
| Flow cytometer channel | FITC | RPE |
| Storage conditions | –20°C | –20°C |
Protocol tips
- Phosphate-containing buffers may interfere with SYBR 14 dye staining
- Semen dilutions of 1:10 (goat) to 1:40 (bovine) result in acceptable cell densities
- Dye stock solutions should be stored frozen
Bovine sperm labeled with the LIVE/DEAD Sperm Viability Kit.