LIVE/DEAD Sperm Viability Kit Imaging Protocol | Thermo Fisher Scientific

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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:

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.