I am studying the anti-tumor effect of Hoechst33342 dye on A-375 cells. However, when I added 75uM of the dye to the plated cells, the dye seem to precipitate and I get close to 100% cell death. Is this normal? If not what can I do to avoid this precipitation? (the stock Hoechst33342 solution is prepared in distilled autoclave water)
75uM is too much. A 10nM final concentration is good enough to kill cells upon longer incubations such as 24 hours. You may try a dose response from 1nM to 1uM to choose an optimum concentration (final). See my paper Figure-7b.
https://www.researchgate.net/publication/233742776_Blebbishields_the_Emergency_Program_for_Cancer_Stem_Cells_Sphere_Formation_and_Tumorigenesis_after_Apoptosis
Article Blebbishields, the Emergency Program for Cancer Stem Cells: ...
Waaay too much.
I use 5-10 uM for 30-60 min. It is quite toxic to some cell types and potentiates may toxins bacuse it shares xenobiotic drug pumps.
The BEST way to use Hoechst33342 dye is to make up a stock in DMSO at 1/125 of 10 uM and place 2ul in the wells of a fresh, clean plate.
Get your plate with the cells growing and remove 100 ul from each well, transfering to the plate with the DMSO/Hoe. Then put the 100 ul back. This way you know the Hoe is dispersed and you don't have a sudden DMSO/Hoe shoce to the cells were you are adding the dye/DMSO.
You're using too much. I use it at 1:10000 from our stock (10uM maybe?) for 1 hour in live cells and don't have a problem. It's super potent so you don't need much of it.
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