Hello all,
I am using Sytox green dye for dead cell counting using a Live cell imaging instrument. As this is a non-permeable dye for live cells, I expect to get a very low count in untreated cells (Background). When I treat the cells with different cell death inducers, it should give high counts eventually. But many times, when I add Sytox green to the wells containing untreated cells, immediately after that, I get a very high count. Sometimes the count goes down after few hours, sometimes it stays the same. I am using around 100 nM Sytox green concentration. Cells are macrophages. Could you please help me regarding this if anyone has encountered such problems and have any solutions. I am using 12 well tissue culture plates, around 1 million cells per well.
Thanks and regards,
Prabuddha
I have not worked with macrophages, but I think you might need to try a different solvent for Syntox green.
I am using DMSO for the main stock as recommended. Should I use aqueous based solvents for the dilution to lower stock?
Yes, DMSO can be cytotoxic. You must try and aqueous solvent for dilutions.
I have thought about it. But usually what we do is, we make a 50uM DMSO stock and then dilute it in media to a make a stock of 0.1-0.05 uM. The DMSO % goes down to 0.2-0.1%. At this level, DMSO is not toxic to cells. Also, it gives high background sometimes, with some cells. Primary macrophages, sometimes. Immortalised macrophages, almost all the time. Can’t find any relevance. If you have any reference protocol from any article, could you please share with me.
Yes, 0.2-0.1% should not pose any problems, maybe it's the type of cells you're working with. I usually work with plant cells so I cannot be sure. But it's possible that macrophages are naturally taking up the dye from the media to digest it. Sorry I cannot help any further. Hope you find a solution!
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