We use a 1mg/ml stock solution of JC-1 in DMSO. We add this to the medium to achieve a final concentration of 5ug/ml. However, we notice a lot of precipitates of JC-1 in the well, even after 3min vortex. These precipitates stick to the cells and very difficult to wash, and hinder visualization under the confocal microscope. Any idea how to avoid that please?
Place the JC-1 first into a dry plastic container such as a 1.5 mL or 15 mL tube. Then add medium to it quickly in order to get most of it into solution; I recommend this be a 2-10x stock. This will prevent a lot of the precipitation and the solution will be mostly pink. However, there will still be precipitate here at the microscopic level. It is best to finish the preparation by using a probe sonicator, after which no precipitate will be visible even at the microscopic level. After incubating the cells with JC-1 it is best to wash the cells at least once.
Thanks for your answer Jordan. Did you try the sonication before? Can I use a sonication bath instead you think? and how long shall I expose the solution to sonication, suppose I will prepare 1 mL?
The sonication needs to be after adding to aqueous solution because it needs to disrupt the precipitate that forms. In my hands, probe sonication is required. Maybe long duration bath sonication could work, but I'm doubtful.
Jordan,
Is it possible to filter or centrifuge the sonicated probe solution to remove precipitate?
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