LIVE/DEAD Sperm Viability Kit Flow Cytometry Protocol

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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:

Fluorescence microscopy. For a fluorescence microscopy protocol, see LIVE/DEAD Sperm Viability Kit Imaging Protocol

You will need the following for this protocol:

Cells growing in culture
LIVE/DEAD Sperm Viability Kit (Cat. No. L7011)
Flow cytometer
Phosphate-buffered saline (PBS) (Cat. No. 14190-144)
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 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 14	Propidium iodide
Excitation/Emission	488/516 nm	535/617 nm
Flow cytometer channel	FITC	RPE
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.