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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

Dilute

1. Dilute semen sample in Live Cell Imaging Solution plus 10% BSA

Add-2

2. Add 900 µL DMSO to SYBR 14 dye to make a stock solution

Add-2

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

Clock-5

4. Incubate for 5–10 minutes at 37°C

Image

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
live-dead-sperm-s000002
Bovine sperm labeled with the LIVE/DEAD Sperm Viability Kit.