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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 flow cytometer

This protocol should not be used for:

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 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 14Propidium iodide
Excitation/Emission488/516 nm535/617 nm
Flow cytometer channelFITCRPE
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.