Hello,

I'm currently trying to optimize SYTOX Green as an extracellular nucleic acid dye for the detection of NETs in vitro. Unfortunately, compared to my positive NETosis control (neutrophils stimulated with PMA), there is also SYTOX Green positivity in my unstimulated negative control.

This is observed over a range of SYTOX Green titres, and I don't fix my cells and analyse them immediately.

SYTOX Green is a cell impermeant nucleic acid dye, so to determine the membrane integrity of my cells I tested SYTOX Green alongside Zombie NIR - the cells are negative for Zombie NIR, indicating they are alive and the membrane is not compromised.

I have also tested the effect of reducing staining time, lower concentrations between 0.0078nM - 1 uM, and keeping the cells at 4°C/on ice to reduce activation or artificial NETosis throughout the isolation and staining period. There is still SYTOX expression in unstimulated neutrophils.

Does anyone have any suggestions for why there may be SYTOX Green positivity in live cells?

Thanks in advance!