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LIVE/DEAD Sperm Viability Kit Imaging Protocol
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 (BSA) (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 | 488/516 nm | 535/617 nm |
| 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.