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 fluorescence microscope
This protocol should not be used for:
- Flow cytometry; for a flow cytometry protocol, see LIVE/DEAD® Sperm Viability Kit Flow Cytometry Protocol
You will need the following for this protocol:
- Cells growing in culture
- LIVE/DEAD® Sperm Viability Kit (Cat. No. L7011)
- Fluorescence microscope with FITC and TRITC filters
- pH 7.4 HEPES buffer, such as Live Cell Imaging Solution (Cat. No. A14291DJ)
- Bovine serum albumin (Cat. No. 15561020)
- Anhydrous DMSO (Cat. No. D12345)
Assay protocol
1. Dilute semen sample in Live Cell Imaging Solution plus 10% BSA
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 a 1 mL sample of diluted semen to label
4. Incubate for 5–10 minutes at 37°C
5. Image cells
Spectral information and storage
| SYBR® 14 | Propidium Iodide | |
|---|---|---|
| Excitation/Emission (in nm) | 488/516 | 535/617 |
| Standard filter set | FITC or GFP | TRITC |
| EVOS® Light Cube | GFP | TRITC |
| 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.
