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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:

You will need the following for this protocol:

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
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.