Earlier I used Hoechst stain to sort polyploid cells but problem is after sorting cell does not grow. Seem to me cell does not like growing with Hoechst stain.
Do you have any other alternative to sort live polyploid cells to do further experiments on them?
Hello Shariful.
Most of FACS machine can separate in the form of live cells these days. Check the machine out first in your institution. Use Hoechst33322 not others. You should use 2~5 ug/ml at a final concentration for 15~20 min at 37C. Make a very high stock solution to reduce toxic effects of the solvent, probably 1,000~2,000x stock solution. At use dilute first in buffer to make 100x stock. Then dilute second in the final buffer of cell suspension to minimize the solvent effect. You do not need the same concentration for microscopy. The machine can detect a very low fluorescence by laser.
Best wishes.
Hi Dr Lee,
Thanks for detail answer.
As I mentioned before, I used Hoechst-33342 ~5ug/ml final. You suggest Hoechst-33322, is it another dye? Is there any other alternative than Hoechst stain? Anybody has better experience with other alternative?
Hi, Shariful.
Yes, it is. It's Hoechst 33322 (Sigma catalog number B2261). Last thing is washing cells with enough medium to remove completely the excessive amount of the dye 2~3 times with 8 ml each to spin them down.
Hoechst dye intercalating A-T bases and it would be gradually diluted in replication and eventually released. However, you could use even 1~2 ug/ml would be enough to get satisfactory separation of cell fractions. Try and determine the least concentration for cell separation in preliminary test.
Some people propose SiR-Hoechst as a less toxic dye.
Regards.
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