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I'm using DRAQ7 and would like to stain for live/dead fixed cells in microscopy. According to the protocol, DRAQ7 will stain all fixed and permeabilized cells in the preparation. How can I then differentiate between my live and dead cells if all are going to be stained? Is there something I can do to change the protocol?
Asked on Aug 15 2011
Thank you for your inquiry. DRAQ7 is a LIVE cell impermeant far-red DNA dye. It can be used to identify permeable cells in a mix of live and dead cells (i.e. the dead cells) – but in a preparation of fixed cells, all of the cells will be permeant and therefore all will be stained with DRAQ7. If you are trying to differentiate between live and dead cells in a sample (before fixation), then you could simply add DRAQ7 prior to fixation and identify (count) the number of DRAQ7 labelled cells – they will be the dead cells. After fixation, the DRAQ7 will enter all of the cells – and you can now get a total count. Subtraction of dead cell count from total count will give the live cell count. There are of course issues here – especially if the same sample cannot be used prior to and after fixation. Another idea might be to use DRAQ7 and DRAQ5 (ab108410) in sequence prior to fixation. Adding DRAQ7 first will give the dead cell count. Adding DRAQ5 will then give the total count – and usefully both dyes are in the far-red (same emission). Subtraction of dead count from total count will give the live count. The sample could then be fixed. I hope that this is helpful. Please let me know if you have any other questions.
Answered on Aug 15 2011