I used NucBlue Live from ThermoFisher (https://www.thermofisher.com/sg/en/home/references/protocols/cell-and-tissue-analysis/protocols/nucblue-live-readyprobes-protocol.html). I found that this stained the nuclei of cells in aggregates of one cancer cell line, but not the nuclei of another type of cancer cell line. For the latter cancer cell line (the one that couldnt be stained), incubating overnight with the dye did not work either.
Hi Sharon, are you labeling live cultures as indicated in the link? If so it may be an issue where the particular cancer cell line is extruding the dye through xenobiotic transporters. Certain cancer stem cells have this characteristic and they have been isolated using exactly their reluctance to uptake the Hoechst dye used in the "NucBlue" (side population sorting). You should be able to test this by blocking dye efflux through addition of verapamil (inhibits xenobiotic transporters) along with the NucBlue. Verapamil method of blocking Hoechst efflux is well described here if you're interested.. Worth a try if you don't get a better explanation. Good luck!Article Hematopoietic Stem Cell Characterization and Isolation
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