Image of SYTOX® Green Nucleic Acid Stain packaging

Dead cell indicator for microscopy

SYTOX® Green Nucleic Acid Stain is a bright, high-affinity nucleic acid stain that easily penetrates cells with compromised plasma membranes but does not cross the membranes of live cells, making it a useful indicator of dead cells within a population. 

This protocol can be used for:

  • Nucleic acid (nuclear) staining in fluorescence microscopy

This protocol should not be used for:

  • Flow cytometry

You will need the following for this protocol:

Protocol

Labeling fixed cells

First, fix and permeabilize cultured cells with a protocol appropriate for your sample.

1. Wash the cells 1–3 times in a phosphate-free buffer as needed.
2. Prepare the SYTOX® Green staining solution by diluting the stock solution 1:30,000 (167 nM) in a phosphate-free buffer.
3. Add sufficient staining solution to cover the cells.
4. Incubate for 15–30 minutes, protected from light. 
5. Remove the staining solution.
6. Wash the cells 2–3 times in a phosphate-free buffer
7. Image the cells.
Spectral information and storage
  SYTOX® Green 
Excitation/Emission (nm) 504/523
Standard filter set GFP
EVOS® Light Cube GFP
Storage conditions ≤–20°C

 

 

 

Protocol tips

  • Warm to room temperature and briefly centrifuge the DMSO solution to the bottom of the vial each time before use.
  • Try multiple dye concentrations in the range from 10 nM to 1 µM to determine the optimal concentration.
  • In general, the best results are obtained in buffers that do not contain phosphate, such as Hank’s Balanced Salt Solution (Cat. No. 14025-092).
  • Treat all nucleic acid binding dyes as potential mutagens and handle with care.

multicolor staining of BPAE cells
Multicolor staining of BPAECs. Mitochondria of BPAECs were stained with MitoTracker® Red CM-H2XRos. The cells were then fixed, permeabilized, RNase-treated, stained with SYTOX® Green Nucleic Acid Stain, and imaged.